Highly sensitive system and method for analysis of troponin

ABSTRACT

The invention provides methods, compositions, kits, and systems for the sensitive detection of cardiac troponin. Such methods, compositions, kits, and systems are useful in diagnosis, prognosis, and determination of methods of treatment in conditions that involve release of cardiac troponin.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.13/798,547 filed on Mar. 13, 2013, now U.S. Pat. No. 9,182,405, which isa continuation of U.S. patent application Ser. No. 13/667,372, filed onNov. 2, 2012, now U.S. Pat. No. 8,535,895, which is a continuation ofU.S. patent application Ser. No. 12/902,182, filed on Oct. 12, 2010, nowU.S. Pat. No. 8,343,728, which is a continuation of U.S. patentapplication Ser. No. 12/062,412, filed Apr. 3, 2008, now U.S. Pat. No.7,838,250, which claims priority under 35 U.S.C. §119 to U.S.Provisional Application 60/914,995, filed Apr. 30, 2007, and U.S.Provisional Application 60/925,402, filed Apr. 19, 2007. Thisapplication is a continuation in part of U.S. patent application Ser.No. 11/784,213 filed on Apr. 4, 2007 which claims priority under 35U.S.C. §119 to U.S. Provisional Application No. 60/789,304, filed Apr.4, 2006, U.S. Provisional Application 60/793,664, filed on Apr. 19,2006, U.S. Provisional Application 60/808,662, filed on May 26, 2006,U.S. Provisional Application No. 60/861,498, filed Nov. 28, 2006, andU.S. Provisional Application No. 60/872,986, filed Dec. 4, 2006, all ofwhich are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Each year in the United States, some six million people present toemergency departments with chest pain. Although only 15% to 20% of thesepatients are ultimately diagnosed with an acute coronary syndrome (ACS),about half are admitted for evaluation. Conversely, 2% of patients withACS are mistakenly discharged. As patients with ACS have a relativelyhigh risk of major adverse cardiovascular events in the short term,there is a clear need for accurate objective tools by which to identifythem.

Currently used markers for cardiac damage suffer disadvantages thatlimit their clinical usefulness. Cardiac enzyme assays have formed thebasis for determining whether or not there is damage to the cardiacmuscle. Unfortunately, the standard creatine kinase-MB (CK-MB) assay isnot reliable in excluding infarction until 10 to 12 hours after theonset of chest pain. Earlier diagnosis would have very specificadvantages with regard to fibrinolytic therapy and triage. Inadditional, more sensitive markers of cardiac toxicity would allowearlier detection of adverse effects of, e.g., drug therapy.

SUMMARY OF THE INVENTION

In one aspect the invention provides methods.

In some embodiments, the invention provides a method for determining thepresence or absence of a single molecule of troponin or a fragment orcomplex thereof in a sample, including i) labeling the molecule,fragment, or complex, if present, with a label; and ii) detecting thepresence or absence of the label, where the detection of the presence ofthe label indicates the presence of the single molecule, fragment, orcomplex of troponin in the sample. In some embodiments of the methods ofthe invention, the troponin is a cardiac isoform of troponin. In someembodiments of the methods of the invention, the troponin can be cardiactroponin I (cTnI) or cardiac troponin C (cTnC). In some embodiments ofthe methods of the invention, the troponin is cTnI. In some embodimentsof the methods of the invention, a single molecule of troponin can bedetected at a limit of detection of less than about 100 pg/ml. In someembodiments of the methods of the invention, a single molecule ortroponin can be detected at a level of detection of less than about 20pg/ml. In some embodiments of the methods of the invention, the labelincludes a fluorescent moiety. In some embodiments, the fluorescentmoiety is capable of emitting at least about 200 photons when simulatedby a laser emitting light at the excitation wavelength of the moiety,where the laser is focused on a spot not less than about 5 microns indiameter that contains the moiety, and where the total energy directedat the spot by the laser is no more than about 3 microJoules. In someembodiments of the methods of the invention, the fluorescent moietyincludes a molecule that contains at least one substituted indolium ringsystem in which the substituent on the 3-carbon of the indolium ringcontains a chemically reactive group or a conjugated substance group. Insome embodiments of the methods of the invention, the fluorescent moietyincludes a dye. Examples of dyes include, but are not limited to,ALEXA-FLUOR® 488, ALEXA-FLUOR® 532, ALEXA-FLUOR® 647, ALEXA-FLUOR® 680and ALEXA-FLUOR® 700. In some embodiments of the methods of theinvention, the fluorescent moiety includes ALEXA-FLUOR® 647. In someembodiments, the fluorescent moiety includes a molecule that contains atleast one substituted indolium ring system in which the substituent onthe 3-carbon of the indolium ring contains a chemically reactive groupor a conjugated substance group. In some embodiments of the methods ofthe invention, the label further includes a binding partner for thetroponin molecule, fragment, or complex. In some embodiments of themethods of the invention, the binding partner includes an antibodyspecific to the troponin molecule, fragment, or complex. In someembodiments of the methods of the invention, the antibody is specific toa specific region of the troponin molecule. In some embodiments of themethods of the invention, the antibody is specific to a regioncomprising amino acids 27-41 of cardiac troponin I. In some embodimentsof the methods of the invention, the antibody can be a polyclonalantibody. In some embodiments of the methods of the invention, theantibody is a monoclonal antibody. In some embodiments of the methods ofthe invention, the methods further include capturing troponin ortroponin complex on a solid support. In some embodiments of the methodsof the invention, the solid support can be a microtiter plate orparamagnetic beads. In some embodiments of the methods of the invention,the solid support includes a capture partner specific for the troponinor troponin complex that is attached to the solid support. In someembodiments of the methods of the invention, the attachment of thecapture partner to the solid support is noncovalent. In some embodimentsof the methods of the invention, the attachment of the capture partnerto the solid support is covalent. In some embodiments of the methods ofthe invention, the covalent attachment of the capture partner is suchthat the capture partner is attached to the solid support in a specificorientation. In some embodiments of the methods of the invention, thespecific orientation serves to maximize specific binding of the troponinor troponin complex to the capture partner. In some embodiments of themethods of the invention, the capture partner comprises an antibody. Insome embodiments of the methods of the invention, the antibody is amonoclonal antibody. In some embodiments of the methods of theinvention, antibody is specific to amino acids 87-91 of cardiac troponinI. In some embodiments of the methods of the invention, the antibody isspecific to amino acids 41-49 of cardiac troponin I. In some embodimentsof the methods of the invention, the sample is a blood, serum, or plasmasample. In some embodiments of the methods of the invention, the sampleis a serum sample. In some embodiments of the methods of the invention,the label include a fluorescent moiety, and step ii) includes passingthe label through a single molecule detector. In some embodiments of themethods of the invention, the single molecule detector include: a) anelectromagnetic radiation source for stimulating the fluorescent moiety;b) a capillary flow cell for passing the fluorescent moiety; c) a sourceof motive force for moving the fluorescent moiety in the capillary flowcell; d) an interrogation space defined within the capillary flow cellfor receiving electromagnetic radiation emitted from the electromagneticsource; e) an electromagnetic radiation detector operably connected tothe interrogation space for measuring an electromagnetic characteristicof the stimulated fluorescent moiety; and f) a microscope objective lenssituated between the interrogation space and the detector, where thelens is a high numerical aperture lens.

In some embodiments, the invention provides a method for determining adiagnosis, prognosis, or method of treatment in an individual thatincludes: i) determining a concentration of cardiac troponin in a sampleor determining the concentrations of cardiac troponin in a series ofsamples from the individual, where the concentration is determined by acardiac troponin assay with a limit of detection for the cardiactroponin in the sample of less than about 50 pg/ml; and ii) determininga diagnosis, prognosis, or method of treatment in the individual, basedon the concentration in the sample, or on the concentrations in theseries of samples. In some embodiments of the methods of the invention,step ii) includes an analysis such as comparing the concentration orseries of concentrations to a normal value for the concentration,comparing the concentration or series of concentrations to apredetermined threshold level, comparing the concentration or series ofconcentrations to a baseline value, and determining a rate of change ofconcentration for the series of concentrations. In some embodiments ofthe methods of the invention, step ii) includes comparing theconcentration of troponin in the sample with a predetermined thresholdconcentration, and determining a diagnosis, prognosis, or method oftreatment if the sample concentration is greater than the thresholdlevel. In some embodiments of the methods of the invention, thethreshold concentration is determined by determining a the 99thpercentile concentration of troponin in a group of normal individuals,and setting the threshold concentration at the 99th percentileconcentration. In some embodiments of the methods of the invention, atleast one sample is taken during or after a cardiac stress test. In someembodiments of the methods of the invention, the cardiac troponin isselected from the group consisting of cardiac troponin I and cardiactroponin T. In some embodiments of the methods of the invention, thecardiac troponin is cardiac troponin I. In some embodiments of themethods of the invention, the concentration of cardiac troponin is aconcentration of total cardiac troponin. In some embodiments of themethods of the invention, the concentration of cardiac troponin is aconcentration of a cardiac troponin complex, cardiac troponin fragment,phosphorylated cardiac troponin, oxidized cardiac troponin, or acombination thereof. In some embodiments of the methods of theinvention, the concentration of cardiac troponin is compared to totalcardiac troponin. In some embodiments of the methods of the invention,the diagnosis, prognosis, or method of treatment is a diagnosis,prognosis, or method of treatment of myocardial infarct. In someembodiments of the methods of the invention, the diagnosis, prognosis,or method of treatment comprises risk stratification for level of riskof myocardial infarct. In some embodiments of the methods of theinvention, the concentration or series of concentrations is determinedat or near the time the individual presents to a health professionalwith one or more symptoms indicative of myocardial ischemia or infarctor the possibility thereof. In some embodiments, the one or moresymptoms can be chest pain, chest pressure, arm pain, abnormal EKG,abnormal enzyme levels, or shortness of breath. In some embodiments, theconcentration is determined by a method that includes detecting singlemolecules of troponin, or complexes or fragments thereof. In someembodiments, the methods of the invention involve labeling troponin or atroponin complex with a label that comprises a fluorescent moiety. Insome embodiments of the methods of the invention, the fluorescent moietyis capable of emitting at least about 200 photons when simulated by alaser emitting light at the excitation wavelength of the moiety, wherethe laser is focused on a spot 5 microns in diameter that contains themoiety, and where the total energy directed at the spot by the laser isno more than about 3 microJoules. In some embodiments of the methods ofthe invention, the fluorescent moiety includes a molecule that containsat least one substituted indolium ring system in which the substituenton the 3-carbon of the indolium ring contains a chemically reactivegroup or a conjugated substance group. In some embodiments of themethods of the invention, the fluorescent moiety includes a dye selectedfrom the group consisting of ALEXA-FLUOR® 488, ALEXA-FLUOR® 532,ALEXA-FLUOR® 647, ALEXA-FLUOR®680 or ALEXA-FLUOR® 700. In someembodiments of the methods of the invention, the fluorescent moietycomprises ALEXA-FLUOR® 647. In some embodiments of the methods of theinvention, the label further comprises a binding partner for thetroponin. In some embodiments, the binding partner comprises an antibodyspecific to the troponin. In some embodiments, the antibody is apolyclonal antibody. In some embodiments of the methods of theinvention, the methods further include capturing troponin or troponincomplex on a solid support. In some embodiments of the methods of theinvention, the solid support can be a microtiter plate or paramagneticbeads. In some embodiments of the methods of the invention, the solidsupport includes a capture partner specific for the troponin or troponincomplex that is attached to the solid support. In some embodiments ofthe methods of the invention, the attachment of the capture partner tothe solid support is noncovalent. In some embodiments of the methods ofthe invention, the attachment of the capture partner to the solidsupport is covalent. In some embodiments of the methods of theinvention, the covalent attachment of the capture partner is such thatthe capture partner is attached to the solid support in a specificorientation. In some embodiments of the methods of the invention, thespecific orientation serves to maximize specific binding of the troponinor troponin complex to the capture partner. In some embodiments of themethods of the invention, step i) further involves assessing anotherindicator for the individual, and step ii) involves determining adiagnosis, prognosis, or method of treatment in the individual, based onthe concentration of troponin and the assessment of the other indicatorof the non-troponin marker in the sample, or on the concentrations inthe series of samples. In some embodiments, the other indicator is aclinical indicator of myocardial ischemia or infarct. In someembodiments, the other indicator is the concentration of one or morenon-troponin markers in the sample or the series of samples. In someembodiments of the methods of the invention, the one or more markers aremarkers of cardiac ischemia, or markers of inflammation and of plaqueinstability. In some embodiments, the one or more markers of cardiacischemia can be creatine kinase (CK) and its myocardial fraction CKmyocardial band (MB), aspartate aminotransferase, lactate dehydrogenase(LDH), α-hydroxybutyrate dehaydrogenase, myoglobin, glutamateoxaloacetate transaminase, glycogen phosphorylase BB, unbound free fattyacids, heart fatty acid binding protein (H-FABP), ischemia-modifiedalbumin, myosin light chain 1, or myosin light chain 2 In someembodiments of the methods of the invention, the one or more markersinclude one or more specific markers of myocardial injury. In someembodiments of the methods of the invention, the diagnosis, prognosis,or method of treatment is a diagnosis, prognosis, or method of treatmentof a condition that is not myocardial infarct. In some embodiments, thecondition is cardiac toxicity. In some embodiments, the cardiac toxicityis associated with the administration of a drug to the individual. Insome embodiments of the methods of the invention, the condition isselected from the group consisting of acute rheumatic fever,amyloidosis, cardiac trauma (including contusion, ablation, pacing,firing, cardioversion, catheterization and cardiac surgery), reperfusioninjury, congestive heart failure, end-stage renal failure, glycogenstorage disease type II (Pompe's disease), heart transplantation,haeomoglobinopathy with transfusion haemosiderosis, hypertension,including gestational hypertension, hypotension, often with arrhythmias,hypothyroidism, myocarditis, pericarditis, post-operative non-cardiacsurgery, pulmonary embolism, and sepsis.

In some embodiments, provided is a method of classifying an individualhaving one or more symptoms of cardiac infarct comprising: i) obtaininga sample from said individual, ii) detecting the level of troponin inthe sample and iii) if the level of troponin is above 10-fold of the99th percentile, taking a first action with respect to the individualand if the level of troponin is below 10-fold of the 99th percentiletaking a second action. In some embodiments, the first action is takenif patient has about a 3-fold to about a 10-fold increase relative tothe 99th percentile cutoff. In some embodiments, the first action istaken if patient has about a 4-fold to about a 5-fold increase relativeto the 99th percentile cutoff. In some embodiments, the first action istaken if patient has about a 6-fold to about a 7-fold increase relativeto the 99th percentile cutoff. In some embodiments, the first action istaken if patient has about an 8-fold to about a 10-fold increaserelative to the 99th percentile cutoff. In some embodiments, the firstaction is taken if patient has about a 1.5-fold to about a 2-foldincrease. In some embodiments, the first action is taken if patient hasabout a 2-fold to about a 2.5-fold increase. In some embodiments, thefirst action is taken if patient has about a 2.5-fold to about a 3-foldincrease. In some embodiments, the first action is taken if patient hasabout a 3-fold to about a 3.5-fold increase. In some embodiments, thefirst action is taken if patient has about a 3.5-fold to about a 4-foldincrease. In some embodiments, the first action is taken if patient hasabout a 4-fold to about a 4.5-fold increase. In some embodiments, thefirst action is taken if patient has about a 4.5-fold to about a 5-foldincrease. In some embodiments, the first action is taken if patient hasabout a 5-fold to about a 5.5-fold increase. In some embodiments, thefirst action is taken if patient has about a 5.5-fold to about a 6-foldincrease. In some embodiments, the first action is taken if patient hasabout a 6-fold to about a 6.5-fold increase. In some embodiments, thefirst action is taken if patient has about a 6.5-fold to about a 7-foldincrease. In some embodiments, the first action is taken if patient hasabout a 7-fold to about a 7.5-fold increase. In some embodiments, thefirst action is taken if patient has about a 7.5-fold to about an 8-foldincrease. In some embodiments, the first action is taken if patient hasabout a 8-fold to about a 8.5-fold increase. In some embodiments, thefirst action is taken if patient has about an 8.5-fold to about a 9-foldincrease. In some embodiments, the first action is taken if patient hasabout a 9-fold to about a 9.5-fold increase. In some embodiments, thefirst action is taken if patient has about a 9.5-fold to about a 10-foldincrease. In some embodiments the first action is taken if the increaseis more than about 2-fold. In some embodiments the first action is takenif the increase is more than about 3-fold. In some embodiments the firstaction is taken if the increase is more than about 4-fold. In someembodiments the first action is taken if the increase is more than about5-fold. In some embodiments the first action is taken if the increase ismore than about 6-fold. In some embodiments the first action is taken ifthe increase is more than about 7-fold. In some embodiments the firstaction is taken if the increase is more than about 8-fold. In someembodiments the first action is taken if the increase is more than about9-fold. In some embodiments the first action is taken if the increase ismore than about 10-fold.

In some embodiments, the individual is a patient being evaluated for apossible cardiac event. In such a case, the first action can beadmission of the individual to a hospital, or other appropriate clinicalaction for treatment of the cardiac event. The second action can be tohold the individual for a period of time for further observation. Duringthe time the individual is held a series of samples are taken from saidpatient and the level of troponin in each sample detected. In someembodiments, the interval between samples is less than about 4, 3, 2, 1,0.5 hours apart. In some embodiments, the sample interval is less thanabout 4 hours apart. In some embodiments, the sample interval is about 1hour. In some embodiments, the sample interval is about 2 hours. It willbe recognized that circumstances in the clinic dictate sample intervalsand some degree of variation is acceptable and is encompassed within theinvention, as long as results may be reliably interpreted. In someembodiments, in addition to the level of troponin detected in eachsample, the rate of change in the level of troponin over two or moresamples in the series of samples is detected. In some embodiments, thechange in the level of troponin is a decrease. In some embodiments, thechange in the level of troponin is an increase. A decision can be maderegarding a course of action for said individual based on said rate ofchange, e.g., if the rate of change in troponin values exceeds apredetermined upper reference rate of change value, the decision is toadmit the individual. It will be recognized that other clinical factorswell-known to those of skill in the art can be used to modify thedecision based on troponin levels, e.g., if other clinicalmanifestations of acute myocardial infarct are present, such as abnormalEKG, abnormal enzyme levels, physical symptoms, and the like, then thethreshold for troponin that leads to an admission is suitably decreased.The decision to take a first action, e.g., to admit a patient, can alsobe made if one or more spikes in the level of troponin are seen fromsample to sample. The spike can be any increase in the level of troponindetected compared to one or more samples wherein the increase iscompared to a threshold level. For example, if the threshold level isdetermined to be 10 pg/ml and if in one of the sample of the series ofsamples the troponin level detected is 15 pg/ml, the patient isadmitted. In some embodiments, the threshold level is determined from areference population. In some embodiments, the threshold level is set ata level for an individual, e.g., the initial level of troponin detectedfrom the first sample taken from the patient. In these embodiments, arelative increase indicates a spike, no matter what the absolute valuesare. In some embodiments, a spike is determined when the troponin levelincreases 2, 3, 4, 5, 6, 7, 8, 9 10, or more than 10-fold above areference value for the individual, e.g., a value set at the troponinlevel in the first sample taken from the individual. Alternatively, aspike in the level of troponin can be seen when comparing troponinlevels between samples from the individual. In such a case, no matterwhat the value of the level of troponin detected, the patient isadmitted if the value increases by more than a certain amount, e.g., apatient is admitted when the level of troponin increases by 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10 pg/ml as compared to one or moreneighboring samples. In some embodiments, the change in the level oftroponin indicates an acute cardiovascular disease. In some embodiments,the change in the level of troponin indicates a chronic cardiovasculardisease. In some embodiments, the change in the level of troponinindicates cardiotoxicity.

In another aspect the invention includes compositions.

In some embodiments the invention includes a composition for thedetection of a troponin isoform including a binding partner to thetroponin isoform attached to a fluorescent moiety, where the fluorescentmoiety is capable of emitting at least about 200 photons when simulatedby a laser emitting light at the excitation wavelength of the moiety,where the laser is focused on a spot not less than about 5 microns indiameter that contains the moiety, and where the total energy directedat the spot by the laser is no more than about 3 microJoules. In someembodiments of the compositions of the invention, the binding partnercomprises an antibody to the troponin isoform. In some embodiments, theantibody is a polyclonal antibody. In some embodiments, the antibody isa monoclonal antibody. In some embodiments, the troponin isoform is acardiac isoform. In some embodiments, the cardiac isoform is selectedfrom the group consisting of cTnI and cTnT. In some embodiments, thecardiac isoform is cTnI. In some embodiments, the antibody is specificto a specific region of the troponin molecule. In some embodiments, theantibody is specific to a region comprising amino acids 27-41 of cardiactroponin I. In some embodiments of the compositions of the invention,the fluorescent moiety comprises a molecule that comprises at least onesubstituted indolium ring system in which the substituent on the3-carbon of the indolium ring contains a chemically reactive group or aconjugated substance group. In some embodiments, the fluorescent moietyincludes a dye that can be ALEXA-FLUOR® 488, ALEXA-FLUOR® 532,ALEXA-FLUOR® 647, ALEXA-FLUOR® 680 or ALEXA-FLUOR® 700. In someembodiments, the fluorescent moiety comprises ALEXA-FLUOR® 647.

In some embodiments the invention involves a composition comprising aset of standards for the determination of a concentration of a cardiactroponin, where at least one of the standards is at a concentration ofcardiac troponin less than about 10 pg/ml.

In another embodiment, provided herein is a composition comprising alabel for cardiac troponin comprising a detection binding partner forcardiac troponin I, wherein the detection binding partner is capable ofcross-reacting with cardiac troponin I from at least two species, and afluorescent moiety, wherein said moiety is capable of emitting at leastabout 200 photons when simulated by a laser emitting light at theexcitation wavelength of the moiety, wherein the laser is focused on aspot not less than about 5 microns in diameter that contains the moiety,and wherein the total energy directed at the spot by the laser is nomore than about 3 microJoules. In some embodiments, the detectionbinding partner is capable of reacting with cardiac troponin I from atleast two species selected from the group consisting of human, monkey,dog, and rat. In some embodiments, the detection binding partner iscapable of reacting with cardiac troponin I from human, monkey, dog, andrat. The detection binding partner can be an antibody. In someembodiments, the composition further comprises a capture binding partnerfor cardiac troponin I, wherein the capture binding partner is capableof cross-reacting with cardiac troponin I from at least two species. Thecapture binding partner can be capable of reacting with cardiac troponinI from at least two species selected from the group consisting of human,monkey, dog, and rat. In some embodiments, the capture binding partneris capable of reacting with cardiac troponin I from human, monkey, dog,and rat. In some embodiments the capture binding partner is an antibody.

In some embodiments the invention involves a kit containing acomposition including an antibody to cardiac troponin attached to afluorescent dye moiety, where the moiety is capable of emitting at leastabout 200 photons when simulated by a laser emitting light at theexcitation wavelength of the moiety, where the laser is focused on aspot not less than about 5 microns in diameter that contains the moiety,and where the total energy directed at the spot by the laser is no morethan about 3 microJoules, where the composition is packaged in suitablepackaging. In some embodiments of the kits of the invention, the cardiactroponin is cardiac troponin I or cardiac troponin T. In someembodiments, the cardiac troponin is cardiac troponin I. In someembodiments of the kits of the invention, the kits further includeinstructions. In some embodiments of the kits of the invention, the kitsfurther include a composition containing a capture antibody for thecardiac troponin I attached to a solid support. In some embodiments, thesolid support comprises a microtiter plate or paramagneticmicroparticles. In some embodiments of the kits of the invention, thekits further include a component selected from the group consisting ofwash buffer, assay buffer, elution buffer, and calibrator diluent. Insome embodiments of the kits of the invention, further include astandard for the cardiac troponin.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A and 1B. Schematic diagram of the arrangement of the componentsof a single particle analyzer. FIG. 1A shows an analyzer that includesone electromagnetic source and one electromagnetic detector; FIG. 1Bshows an analyzer that includes two electromagnetic sources and oneelectromagnetic detectors.

FIGS. 2A and 2B. Schematic diagrams of a capillary flow cell for asingle particle analyzer. FIG. 2A shows the flow cell of an analyzerthat includes one electromagnetic source; and FIG. 2B shows the flowcell of an analyzer that includes two electromagnetic sources.

FIGS. 3A and 3B. Schematic diagrams showing the conventional (A) andconfocal (B) positioning of laser and detector optics of a singleparticle analyzer. FIG. 3A shows the arrangement for an analyzer thathas one electromagnetic source and one electromagnetic detector; FIG. 3Bshows the arrangement for an analyzer that has two electromagneticsources and two electromagnetic detectors.

FIG. 4. Linearized standard curve for the range concentrations of cTnI.

FIG. 5. Biological threshold (cutoff concentration) for cTnI is at acTnI concentration of 7 pg/ml, as established at the 99th percentilewith a corresponding CV of 10%.

FIG. 6. Correlation of assay results of cTnI determined using theanalyzer system of the invention with standard measurements provided bythe National Institute of Standards and Technology (R2=0.9999).

FIG. 7. Detection of cTnI in serial serum samples from patients whopresented at the emergency room with chest pain. The measurements madewith the analyzer system of the invention were compared to measurementsmade with a commercially available assay.

FIG. 8. Distribution of normal biological concentrations of cTnI (NoIschemia) and concentrations of cTnI in serum samples from patientspresenting with chest pain.

FIG. 9. Table showing cross-reactivity of capture/detection antibodypair across human, monkey, dog, and rat.

FIG. 10. Table showing endogenous serum levels of cTnI in human, monkey,dog, and rat.

FIG. 11. Linear regression analysis of back interpolated values from atypical human IL-6 standard Curve

FIGS. 12A and 12B. Graph showing interassay precision; backinterpolation of cTni standard curves generated over 8 consecutive assayruns. FIG. 12A shows the full range of values; FIG. 12B shows the lowend range of quantification.

FIG. 13. Frequency distribution of cTnI in lithium heparin plasmaspecimens obtained from 100 different blood donors.

FIGS. 14A and 14B. Distribution of results of biological variation forcardiac troponin. FIG. 14A shows the short-term biological variation forcardiac troponin; FIG. 14B shows the long-term biological variation forcardiac troponin.

FIG. 15. Graph showing concentrations of cTnI (ng/L) in matched serumand plasma specimens obtained from 20 human donors of mixed age and sexSamples were run in triplicate and results for each sample are presentedas the mean+/−standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

Summary

-   I. Introduction-   II. Cardiac Troponin-   III. Labels for Cardiac Troponin    -   A. Binding partners for troponin        -   1. Antibodies        -   2. Cross-reacting antibodies    -   B. Fluorescent Moieties To Be Used With Binding Partners        -   1. Dyes        -   2 Quantum dots    -   C. Binding Partner-Fluorescent Moiety Compositions-   IV. Highly Sensitive Analysis of Cardiac Troponin    -   A. Sample    -   B. Sample preparation    -   C. Detection of troponin and determination of concentration-   V. Instruments and Systems Suitable for Highly Sensitive Analysis of    Troponin    -   A. Apparatus/System    -   B. Single Particle Analyzer        -   1. Electromagnetic Radiation Source        -   2. Capillary Flow Cell        -   3. Motive Force        -   4. Detectors    -   C. Sampling System    -   D. Sample preparation system    -   E. Sample recovery-   VI. Methods Using Highly Sensitive Analysis of Cardiac Troponin    -   A. Samples    -   B. Determination of diagnosis, prognosis, or method of treatment        -   1. Acute myocardial infarct        -   2. Conditions other than AMI            -   a. Cardiac toxicity    -   C. Business Methods-   VII. Compositions-   VIII. Kits    I. Introduction

The invention provides compositions and methods for the highly sensitivedetection of troponin, e.g., cardiac troponin. The release into theblood of the cardiac isoforms of troponin, which are unique to cardiacmuscle (cardiac troponin I and/or T) is indicative of damage to cardiacmuscle, and provides the basis for their use as diagnostic or prognosticmarkers, or to aid in determination of treatment.

The troponin complex in muscle consists of troponin I, C and T. TroponinC exists as two isoforms, one from cardiac and slow-twitch muscle andone from fast-twitch muscle; because it is found in virtually allstriated muscle, its use as a specific marker is limited. In contrast,troponin I and T are expressed as different isoforms in slow-twitch,fast-twitch and cardiac muscle. The unique cardiac isoforms of troponinI and T allow them to be distinguished immunologically from the othertroponins of skeletal muscle. Therefore, the release into the blood ofcardiac troponin I and T is indicative of damage to cardiac muscle, andprovides the basis for their use as diagnostic or prognostic markers, orto aid in determination of treatment.

Currently used markers for cardiac damage suffer disadvantages thatlimit their clinical usefulness. Cardiac enzyme assays have formed thebasis for determining whether or not there is damage to the cardiacmuscle. Unfortunately, the standard creatine kinase-MB (CK-MB) assay isnot reliable in excluding infarction until 10 to 12 hours after theonset of chest pain. Earlier diagnosis would have very specificadvantages with regard to fibrinolytic therapy and triage.

Because the level of troponin found in the circulation of healthyindividuals is very low, and cardiac specific troponins do not arisefrom extra-cardiac sources, the troponins are very sensitive andspecific markers of cardiac injury. In addition to cardiac infarct, anumber of other conditions can cause damage to the heart muscle, andearly detection of such damage would prove useful to clinicians.However, present methods of detection and quantitation of cardiactroponin do not possess sufficient sensitivity to detect the release ofcardiac troponin into the blood until levels have reached abnormallyhigh concentrations, e.g., 0.1 ng/ml or greater.

The methods and compositions of the invention thus include methods andcompositions for the highly sensitive detection and quantitation ofcardiac troponin, and compositions and methods for diagnosis, prognosis,and/or determination of treatment based on such highly sensitivedetection and quantitation.

II. Cardiac Troponin

When the two unique forms of cardiac troponin, cardiac troponin I (cTnI)and cardiac troponin (cTnT) are released into the blood from cardiacmuscle, several species of each may exist in the blood. These includevarious complexes of the two forms, with each other and/or with cardiactroponin C (cTnC). In addition, the two forms are subject to virtuallyimmediate proteolytic degradation, resulting in a variety of fragments.Also, various phosphorylated and oxidized forms of the troponins mayexist in the blood. See, e.g., U.S. Pat. No. 6,991,907, incorporated byreference herein in its entirety. Unless otherwise specified, “cardiactroponin,” as used herein, encompasses all forms of cardiac troponin,including

In some embodiments, the invention provides methods and compositions forthe detection and/or determination of concentration of total cardiactroponin, i.e., the sum of all or a substantial portion of the cardiactroponin in a sample, e.g., blood, serum or plasma sample, whether it isfree, complexed, a proteotlytic fragment, phosphorylated, oxidized, orotherwise modified. In some embodiments, the cardiac troponin is cTnI,in others, it is cTnT, and in still other embodiments, the cardiactroponin is cTnI and cTnT. It will be appreciated that an absolute totalmeasurement need not be achieved, as long as a consistent proportion ofthe total is determined, which can be compared to standard values. Itwill also be appreciated that if a form of troponin is a minorconstituent of the total, absence or low levels of detection of thatform will not appreciably affect measures of total troponin. Thus, asused herein, “total cardiac troponin” refers to a measurement that isintended to measure all or substantially all forms of a particularcardiac troponin, e.g., all cTnI, or all cTnT, in a sample, where thesample-to-sample consistency is such that clinically relevantconclusions may be drawn from comparisons of samples to standards, orcomparison of one sample to another.

In some embodiments, the invention provides methods and compositions forthe detection and/or determination of concentration of one or more ofthe various forms of troponin in the sample as a separate entity, e.g.,complexed cTnI, free cTnI, muddied cTnI (e.g., oxidized orphosphorylated), or complexed cTnT, free cTnT, muddied cTnT (e.g.,oxidized or phosphorylated), and, typically, can provide a concentrationfor that form in the sample. In the latter embodiments, ratios orabsolute values may be determined for the different entities. Thus, insome embodiments, the invention provides methods of detecting and,typically, determining the concentration of, one or more forms ofcomplexed troponin, or one or more fragments of troponin, or one or moreoxidized or phosphorylated forms of troponin. In some embodiments, morethan one form is detected, and the concentrations of the various formsmay be determined e.g., by performing multiplexed assays on a singlesample for the different entities, or by performing separate assays onaliquots from the same or similar samples. Ratios of concentrations ofthe various forms may be obtained. For example, a ratio of theconcentration of a particular form, e.g., a fragment, complex, ormodified form, of the cardiac troponin to the concentration of totalcardiac troponin, may be determined. These ratios and/or absolute valuescan provide meaningful clinical information. For example the relativeproportion of fragments of cardiac troponin can indicate the length oftime since release into the blood and thus, indirectly, length of timesince, e.g., myocardial infarct. See, e.g., U.S. Pat. No. 6,991,907,incorporated by reference herein in its entirety.

III. Labels for Cardiac Troponin

In some embodiments, the invention provides methods and compositionsthat include labels for the highly sensitive detection and quantitationof cardiac troponin.

One skilled in the art will recognize that many strategies can be usedfor labeling target molecules to enable their detection ordiscrimination in a mixture of particles. The labels may be attached byany known means, including methods that utilize non-specific or specificinteractions of label and target. Labels may provide a detectable signalor affect the mobility of the particle in an electric field. Inaddition, labeling can be accomplished directly or through bindingpartners.

In some embodiments, the label comprises a binding partner to troponinattached to a fluorescent moiety.

A. Binding Partners for Troponin

Any suitable binding partner with the requisite specificity for the formof cardiac troponin to be detected may be used. For example, a bindingpartner specific to all or substantially all forms of cTnI may be usedor a binding partner specific to all or substantially all forms of cTnTmay be used; typically such binding partners bind to a region of thecardiac troponin that is common to all or most of the different formslikely to be found in a sample. In some embodiments, a binding partnerspecific to one or more particular forms of cardiac troponin may beused, e.g., a binding partner to complexed cTnI, free cTnI, muddied cTnI(e.g., oxidized or phosphorylated), or complexed cTnT, free cTnT,muddied cTnT (e.g., oxidized or phosphorylated). Binding partners areknown in the art and include, e.g., aptamers, lectins, and receptors. Auseful and versatile type of binding partner is an antibody.

1. Antibodies

In some embodiments, the binding partner is an antibody specific for acardiac troponin. The term “antibody,” as used herein, is a broad termand is used in its ordinary sense, including, without limitation, torefer to naturally occurring antibodies as well as non-naturallyoccurring antibodies, including, for example, single chain antibodies,chimeric, bifunctional and humanized antibodies, as well asantigen-binding fragments thereof. In some embodiments, the antibody isspecific for cTnI. In some embodiments, the antibody is specific forcTnT. In some embodiments, the label includes antibodies to both cTnIand cTnT. The antibody may be specific to all or substantially all formsthe cardiac troponin; e.g., all or substantially all forms of cTnI, orall or substantially all forms of cTnT. In some embodiments, an antibodyspecific to one or more particular forms of cardiac troponin may beused, e.g., a binding partner to complexed cTnI, free cTnI, muddied cTnI(e.g., oxidized or phosphorylated), or complexed cTnT, free cTnT,muddied cTnT (e.g., oxidized or phosphorylated). Mixtures of antibodiesare also encompassed by the invention, e.g., mixtures of antibodies tocTnI and cTnT, or mixtures of antibodies to the various forms of thetroponin (free, complexed, etc.), or mixtures of mixtures.

It will be appreciated that the choice of epitope or region of troponinto which the antibody is raised will determine its specificity, e.g.,for total troponin, for certain fragments, for complexed troponin, formodified troponin, and the like. In some embodiments, the antibody isspecific to a specific amino acid region of a cardiac troponin. In someembodiments, the antibody is specific to amino acids 27-41 of humancardiac troponin I. Both monoclonal and polyclonal antibodies are usefulas binding partners. In some embodiments, the antibody is a polyclonalantibody. In some embodiments, the antibody is a monoclonal antibody. Insome embodiments, the antibody is a polyclonal antibody specific toamino acids 27-41 of human cardiac troponin I. In some embodiments, thisantibody is not affected by heparin, phosphorylation, oxidation andtroponin complex formation, and does not cross-react with skeletalmuscle troponin I.

Methods for producing antibodies are well-established. The cardiacspecific sequences to troponin I and troponin T are described in FEBSLett. 270, 57-61 (1990) and Genomics 21, 311-316 (1994). One skilled inthe art will recognize that many procedures are available for theproduction of antibodies, for example, as described in Antibodies, ALaboratory Manual, Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art willalso appreciate that binding fragments or Fab fragments which mimicantibodies can also be prepared from genetic information by variousprocedures (Antibody Engineering: A Practical Approach (Borrebaeck, C.,ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920(1992)). Methods for raising antibodies to the various complexed,fragment, phosphorylated, and oxidized forms of the troponins aredisclosed in U.S. Pat. Nos. 5,579,687; 6,991,907; and in US PatentApplication No. 20050164317, which are herein incorporated by referencein their entirety. A synthetic peptide comprised of 14 amino acids whichmimics a cardiac specific sequence of troponin I and methods used toprepare antibodies to the peptide are described in international PatentApplication number PCT/US94/05468. Monoclonal and polyclonal antibodiesto free and complexed cardiac troponins are also commercially available(HyTest, HyTest Ltd., Turku Finland; Abcam Inc., Cambridge, Mass., USA,Life Diagnostics, Inc., West Chester, Pa., USA; Fitzgerald IndustriesInternational, Inc., Concord, Mass. 01742-3049 USA; BiosPacific,Emeryville, Calif.).

In some embodiments, the antibody is a mammalian, e.g., goat polyclonalanti-cTnI antibody. The antibody may be specific to specific regions ofthe cTnI, e.g., amino acids 27-41 of human cardiac troponin I. Capturebinding partners and detection binding partner pairs, e.g., capture anddetection antibody pairs, may be used in embodiments of the invention.Thus, in some embodiments, a heterogeneous assay protocol is used inwhich, typically, two binding partners, e.g., two antibodies, are used.One binding partner is a capture partner, usually immobilized on a solidsupport, and the other binding partner is a detection binding partner,typically with a detectable label attached. In some embodiments, thecapture binding partner member of a pair is an antibody that is specificto all or substantially all forms of cardiac troponin. An example is anantibody, e.g., a monoclonal antibody, specific to free cardiac troponinI (cTnI) a.a. 41-49 and cTnI forming complexes with other troponincomponents. Preferably, this antibody is not affected by heparin,phosphorylation, oxidation and troponin complex formation, and does notcross-react with skeletal muscle troponin I. Thus, it is thought thatthe antibody binds to total cTnI. Another example is a monoclonalantibody, specific to cardiac troponin I (cTnI) a.a. 87-91 and does notcross-react with skeletal muscle troponin I. Such antibodies areavailable from BiosPacific, Emeryville, Calif. Other antibody pairs areknown or can be designed.

Cross-reacting antibodies In some embodiments it is useful to use anantibody that cross-reacts with a variety of species, either as acapture antibody, a detection antibody, or both. Such embodimentsinclude the measurement of drug toxicity by determining, e.g., releaseof cardiac troponin into the blood as a marker of cardiac damage. Across-reacting antibody allows studies of toxicity to be done in onespecies, e.g. a non-human species, and direct transfer of the results tostudies or clinical observations of another species, e.g., humans, usingthe same antibody or antibody pair in the reagents of the assays, thusdecreasing variability between assays. Thus, in some embodiments, one ormore of the antibodies for use as a binding partner to the marker, e.g.,cardiac troponin, such as cardiac troponin I, may be a cross-reactingantibody. In some embodiments, the antibody cross-reacts with themarker, e.g. cardiac troponin, from at least two species selected fromthe group consisting of human, monkey, dog, rat, and mouse. In someembodiments the antibody cross-reacts with the marker e.g. cardiactroponin, from all of the group consisting of human, monkey, dog, rat,and mouse. In some embodiments, the antibody cross-reacts with cardiactroponin, e.g., cardiac troponin I, from human, monkey, dog, and rat.Such a cross-reacting antibody may be used in any suitable embodiment asdescribed herein. For example, a cross-reacting antibody pair may beused wherein each member of the pair (i.e., capture and detectionantibody) cross reacts across species with cardiac troponin I. Thecapture antibody may be immobilized on a solid support. In someembodiments, the capture antibody is immobilized on a paramagneticmicroparticle, such as described in Example 5. An example of resultsobtained from a cross-reacting antibody pair for human, monkey, dog, andrat is given in Example 6.

B. Fluorescent Moieties to be Used with Binding Partners

In some embodiments, the binding partner, e.g., antibody, is attached toa fluorescent moiety. The fluorescence of the moiety, will be sufficientto allow detection in a single molecule detector, such as the singlemolecule detectors described herein. A “fluorescent moiety,” as thatterm is used herein, includes one or more fluorescent entities whosetotal fluorescence is such that the moiety may be detected in the singlemolecule detectors described herein. Thus, a fluorescent moiety maycomprise a single entity (e.g., a Quantum Dot or fluorescent molecule)or a plurality of entities (e.g., a plurality of fluorescent molecules).It will be appreciated that when “moiety,” as that term is used herein,refers to a group of fluorescent entities, e.g., a plurality offluorescent dye molecules, each individual entity may be attached to thebinding partner separately or the entities may be attached together, aslong as the entities as a group provide sufficient fluorescence to bedetected.

Typically, the fluorescence of the moiety involves a combination ofquantum efficiency and lack of photobleaching sufficient that the moietyis detectable above background levels in a single molecule detector,with the consistency necessary for the desired level of detection,accuracy, and precision of the assay. For example, in some embodiments,the fluorescence of the fluorescent moiety is such that it allowsdetection and/or quantitation of troponin at a level of detection ofless than about 10, 5, 4, 3, 2, or 1 pg/ml and with a coefficient ofvariation of less than about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, 1% or less, e.g., about 10% or less, in the instrumentsdescribed herein. In some embodiments, the fluorescence of thefluorescent moiety is such that it allows detection and/or quantitationof troponin at a limit of detection of less than about 5 pg/ml and witha coefficient of variation of less than about 10%, in the instrumentsdescribed herein. “Limit of detection,” as that term is used herein,includes the lowest concentration at which one can identify a sample ascontaining a molecule of the substance of interest, e.g., the firstnon-zero value. It can be defined by the variability of zeros and theslope of the standard curve. For example, the limit of detection of anassay may be determined by running a standard curve, determining thestandard curve zero value, and adding 2 standard deviations to thatvalue. A concentration of the substance of interest that produces asignal equal to this value is the “lower limit of detection”concentration.

Furthermore, the moiety has properties that are consistent with its usein the assay of choice. In some embodiments, the assay is animmunoassay, where the fluorescent moiety is attached to an antibody;the moiety must have properties such that it does not aggregate withother antibodies or proteins, or experiences no more aggregation than isconsistent with the required accuracy and precision of the assay. Insome embodiments, fluorescent moieties that are preferred arefluorescent moieties, e.g., dye molecules that have a combination of 1)high absorption coefficient; 2) high quantum yield; 3) high photostability (low photobleaching); and 4) compatibility with labeling thebiomolecule of interest (e.g., protein) so that it may be analyzed usingthe analyzers and systems of the invention (e.g., does not causeprecipitation of the protein of interest, or precipitation of a proteinto which the moiety has been attached).

Fluorescent moieties, e.g., a single fluorescent dye molecule or aplurality of fluorescent dye molecules, that are useful in someembodiments of the invention may be defined in terms of their photonemission characteristics when stimulated by EM radiation. For example,in some embodiments, the invention utilizes a fluorescent dye moiety,e.g., a single fluorescent dye molecule or a plurality of fluorescentdye molecules, that is capable of emitting an average of at least about10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,350, 400, 500, 600, 700, 800, 900, or 1000, photons when simulated by alaser emitting light at the excitation wavelength of the moiety, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the moiety, and wherein the total energy directedat the spot by the laser is no more than about 3 microJoules. It will beappreciated that the total energy may be achieved by many differentcombinations of power output of the laser and length of time of exposureof the dye moiety. E.g., a laser of a power output of 1 mW may be usedfor 3 ms, 3 mW for 1 ms, 6 mW for 0.5 ms, 12 mW for 0.25 ms, and so on.

In some embodiments, the invention utilizes a fluorescent dye moiety,e.g., a single fluorescent dye molecule or a plurality of fluorescentdye molecules, that is capable of emitting an average of at least about50 photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the moiety, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules. In some embodiments, the invention utilizes afluorescent dye moiety, e.g., a single fluorescent dye molecule or aplurality of fluorescent dye molecules, that is capable of emitting anaverage of at least about 100 photons when simulated by a laser emittinglight at the excitation wavelength of the moiety, where the laser isfocused on a spot of not less than about 5 microns in diameter thatcontains the moiety, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the invention utilizes a fluorescent dye moiety, e.g., a singlefluorescent dye molecule or a plurality of fluorescent dye molecules,that is capable of emitting an average of at least about 150 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe moiety, where the laser is focused on a spot of not less than about5 microns in diameter that contains the moiety, and wherein the totalenergy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the invention utilizes a fluorescentdye moiety, e.g., a single fluorescent dye molecule or a plurality offluorescent dye molecules, that is capable of emitting an average of atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the moiety, where the laser is focused on aspot of not less than about 5 microns in diameter that contains themoiety, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules. In some embodiments, the inventionutilizes a fluorescent dye moiety, e.g., a single fluorescent dyemolecule or a plurality of fluorescent dye molecules, that is capable ofemitting an average of at least about 300 photons when simulated by alaser emitting light at the excitation wavelength of the moiety, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the moiety, and wherein the total energy directedat the spot by the laser is no more than about 3 microJoules. In someembodiments, the invention utilizes a fluorescent dye moiety e.g., asingle fluorescent dye molecule or a plurality of fluorescent dyemolecules, that is capable of emitting an average of at least about 500photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the moiety, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules.

In some embodiments, the fluorescent moiety comprises an average of atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fluorescent entities, e.g.,fluorescent molecules. In some embodiments, the fluorescent moietycomprises an average of no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11 fluorescent entities, e.g., fluorescent molecules. In someembodiments, the fluorescent moiety comprises an average of about 1 to11, or about 2 to 10, or about 2 to 8, or about 2 to 6, or about 2 to 5,or about 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 to 6, orabout 3 to 5, or about 4 to 10, or about 4 to 8, or about 4 to 6, orabout 2, 3, 4, 5, 6, or more than about 6 fluorescent entities. In someembodiments, the fluorescent moiety comprises an average of about 2 to 8fluorescent moieties are attached. In some embodiments, an average ofabout 2 to 6 fluorescent entities. In some embodiments, the fluorescentmoiety comprises an average of about 2 to 4 fluorescent entities. Insome embodiments, the fluorescent moiety comprises an average of about 3to 10 fluorescent entities. In some embodiments, the fluorescent moietycomprises an average of about 3 to 8 fluorescent entities. In someembodiments, the fluorescent moiety comprises an average of about 3 to 6fluorescent entities. By “average” is meant that, in a given sample thatis a representative sample of a group of labels of the invention, wherethe sample contains a plurality of the binding partner-fluorescentmoiety units, the molar ratio of the particular fluorescent entity ofwhich the fluorescent moiety is comprise, to the binding partner, asdetermined by standard analytical methods, corresponds to the number orrange of numbers specified. For example, in embodiments in which thelabel comprises a binding partner that is an antibody and a fluorescentmoiety that comprises a plurality of fluorescent dye molecules of aspecific absorbance, a spectrophometric assay may be used in which asolution of the label is diluted to an appropriate level and theabsorbance at 280 nm is taken to determine the molarity of the protein(antibody) and an absorbance at, e.g., 650 nm (for ALEXA-FLUOR® 647) istaken to determine the molarity of the fluorescent dye molecule. Theratio of the latter molarity to the former represents the average numberof fluorescent entities (dye molecules) in the fluorescent moietyattached to each antibody.

1. Dyes

In some embodiments, the invention utilizes fluorescent moieties thatcomprise fluorescent dye molecules. In some embodiments, the inventionutilizes a fluorescent dye molecule that is capable of emitting anaverage of at least about 50 photons when simulated by a laser emittinglight at the excitation wavelength of the molecule, where the laser isfocused on a spot of not less than about 5 microns in diameter thatcontains the molecule, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the invention utilizes a fluorescent dye molecule that is capable ofemitting an average of at least about 75 photons when simulated by alaser emitting light at the excitation wavelength of the molecule, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the molecule, and wherein the total energydirected at the spot by the laser is no more than about 3 microJoules.In some embodiments, the invention utilizes a fluorescent dye moleculethat is capable of emitting an average of at least about 100 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe molecule, where the laser is focused on a spot of not less thanabout 5 microns in diameter that contains the molecule, and wherein thetotal energy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the invention utilizes a fluorescentdye molecule that is capable of emitting an average of at least about150 photons when simulated by a laser emitting light at the excitationwavelength of the molecule, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the molecule, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules. In some embodiments, the invention utilizes afluorescent dye molecule that is capable of emitting an average of atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the molecule, where the laser is focused on aspot of not less than about 5 microns in diameter that contains themolecule, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules

A non-inclusive list of useful fluorescent entities for use in thefluorescent moieties of the invention is given in Table 1, below. Insome embodiments, the fluorescent entity is selected from the groupconsisting of ALEXA-FLUOR® 488, 532, 647, 700, 750, Fluorescein,B-phycoerythrin, allophycocyanin, PBXL-3, and Qdot 605.

TABLE 1 FLUORESCENT ENTITIES Em Dye Ex (nm) E (M)−1 (nm) Mw Bimane 3805,700 458 282.31 Dapoxyl 373 22,000 551 362.83 Dimethylamino 375 22,000470 344.32 coumarin-4-acetic acid Marina blue 365 19,000 460 367.268-Anilino 372 480 naphthalene-1-sulfonic acid Cascade blue 376 23,000420 607.42 ALEXA-FLUOR ® 405 402 35,000 421 1028.26 Cascade blue 40029,000 420 607.42 Cascade yellow 402 24,000 545 563.54 Pacific blue 41046,000 455 339.21 PyMPO 415 26,000 570 582.41 Alexa 430 433 15,000 539701.75 Atto-425 438 486 NBD 465 22,000 535 391.34 Alexa 488 495 73,000519 643.41 Fluorescein 494 79,000 518 376.32 Oregon Green 488 496 76,000524 509.38 Atto 495 495 522 Cy2 489 150,000 506 713.78 DY-480-XL 50040,000 630 514.60 DY-485-XL 485 20,000 560 502.59 DY-490-XL 486 27,000532 536.58 DY-500-XL 505 90,000 555 596.68 DY-520-XL 520 40,000 664514.60 ALEXA-FLUOR ® 532 531 81,000 554 723.77 BODIPY 530/550 534 77,000554 513.31 6-HEX 535 98,000 556 680.07 6-JOE 522 75,000 550 602.34Rhodamine 6G 525 108,000 555 555.59 Atto-520 520 542 Cy3B 558 130,000572 658.00 ALEXA-FLUOR ® 610 612 138,000 628 ALEXA-FLUOR ® 633 632159,000 647 ca. 1200 ALEXA-FLUOR ® 647 650 250,000 668 ca. 1250 BODIPY630/650 625 101,000 640 660.50 Cy5 649 250,000 670 791.99 ALEXA-FLUOR ®660 663 110,000 690 ALEXA-FLUOR ® 680 679 184,000 702 ALEXA-FLUOR ® 700702 192,000 723 ALEXA-FLUOR ® 750 749 240,000 782 B-phycoerythrin 546,565 2,410,000 575 240,000 R-phycoerythrin 480, 546, 565 1,960,000 578240,000 Allophycocyanin 650 700,000 660 700,000 PBXL-1 545 666 PBXL-3614 662 Atto-tec dyes Name Ex (nm) Em (nm) QY □ (ns) Atto 425 436 4860.9 3.5 Atto 495 495 522 0.45 2.4 Atto 520 520 542 0.9 3.6 Atto 560 561585 0.92 3.4 Atto 590 598 634 0.8 3.7 Atto 610 605 630 0.7 3.3 Atto 655665 690 0.3 1.9 Atto 680 680 702 0.3 1.8 Dyomics Fluors molecular Molarabsorbance* weight# label Ex (nm) [l · mol−1 · cm−1] Em (nm) [g · mol−1]DY-495/5 495 70,000 520 489.47 DY-495/6 495 70,000 520 489.47 DY-495X/5495 70,000 520 525.95 DY-495X/6 495 70,000 520 525.95 DY-505/5 50585,000 530 485.49 DY-505/6 505 85,000 530 485.49 DY-505X/5 505 85,000530 523.97 DY-505X/6 505 85,000 530 523.97 DY-550 553 122,000 578 667.76DY-555 555 100.000 580 636.18 DY-610 609 81.000 629 667.75 DY-615 621200.000 641 578.73 DY-630 636 200.000 657 634.84 DY-631 637 185.000 658736.88 DY-633 637 180.000 657 751.92 DY-635 647 175.000 671 658.86DY-636 645 190.000 671 760.91 DY-650 653 170.000 674 686.92 DY-651 653160.000 678 888.96 DYQ-660 660 117,000 — 668.86 DYQ-661 661 116,000 —770.90 DY-675 674 110.000 699 706.91 DY-676 674 145.000 699 807.95DY-680 690 125.000 709 634.84 DY-681 691 125.000 708 736.88 DY-700 70296.000 723 668.86 DY-701 706 115.000 731 770.90 DY-730 734 185.000 750660.88 DY-731 736 225.000 759 762.92 DY-750 747 240.000 776 712.96DY-751 751 220.000 779 814.99 DY-776 771 147.000 801 834.98 DY-780-OH770 70.000 810 757.34 DY-780-P 770 70.000 810 957.55 DY-781 783 98.000800 762.92 DY-782 782 102.000 800 660.88 EVOblue-10 651 101.440 664389.88 EVOblue-30 652 102.000 672 447.51 Quantum Dots: Qdot 525, 565,585, 605, 655, 705, 800

Suitable dyes for use in the invention include modified carbocyaninedyes. The modification of carbocyanine dyes includes the modification ofan indolium ring of the carbocyanine dye to permit a reactive group orconjugated substance at the number 3 position. The modification of theindolium ring provides dye conjugates that are uniformly andsubstantially more fluorescent on proteins, nucleic acids and otherbiopolymers, than conjugates labeled with structurally similarcarbocyanine dyes bound through the nitrogen atom at the number oneposition. In addition to having more intense fluorescence emission thanstructurally similar dyes at virtually identical wavelengths, anddecreased artifacts in their absorption spectra upon conjugation tobiopolymers, the modified carbocyanine dyes have greater photostabilityand higher absorbance (extinction coefficients) at the wavelengths ofpeak absorbance than the structurally similar dyes. Thus, the modifiedcarbocyanine dyes result in greater sensitivity in assays that use themodified dyes and their conjugates. Preferred modified dyes includecompounds that have at least one substituted indolium ring system inwhich the substituent on the 3-carbon of the indolium ring contains achemically reactive group or a conjugated substance. Other dye compoundsinclude compounds that incorporate an azabenzazolium ring moiety and atleast one sulfonate moiety. The modified carbocyanine dyes that can beused to detect individual particles in various embodiments of theinvention are described in U.S. Pat. No. 6,977,305, which is hereinincorporated by reference in its entirety. Thus, in some embodiments thelabels of the invention utilize a fluorescent dye that includes asubstituted indolium ring system in which the substituent on the3-carbon of the indolium ring contains a chemically reactive group or aconjugated substance group.

In some embodiments, the label comprises a fluorescent moiety thatincludes one or more Alexa dyes (Molecular Probes, Eugene, Oreg.). TheAlexa dyes are disclosed in U.S. Pat. Nos. 6,977,305; 6,974,874;6,130,101; and 6,974,305, which are herein incorporated by reference intheir entirety. Some embodiments of the invention utilize a dye chosenfrom the group consisting of ALEXA-FLUOR® 647, ALEXA-FLUOR® 488,ALEXA-FLUOR® 532, ALEXA-FLUOR® 555, ALEXA-FLUOR® 610, ALEXA-FLUOR® 680,ALEXA-FLUOR® 700, and ALEXA-FLUOR® 750. Some embodiments of theinvention utilize a dye chosen from the group consisting of ALEXA-FLUOR®488, ALEXA-FLUOR® 532, ALEXA-FLUOR® 647, ALEXA-FLUOR® 700 andALEXA-FLUOR® 750. Some embodiments of the invention utilize theALEXA-FLUOR® 647 molecule, which has an absorption maximum between about650 and 660 nm and an emission maximum between about 660 and 670 nm. TheALEXA-FLUOR® 647 dye is used alone or in combination with otherALEXA-FLUOR® dyes.

In addition, currently available organic fluors can be improved byrendering them less hydrophobic by adding hydrophilic groups such aspolyethylene. Alternatively, currently sulfonated organic fluors such asthe ALEXA-FLUOR® 647 dye can be rendered less acidic by making themzwitterionic. Particles such as antibodies that are labeled with themodified fluors are less likely to bind non-specifically to surfaces andproteins in immunoassays, and thus enable assays that have greatersensitivity and lower backgrounds. Methods for modifying and improvingthe properties of fluorescent dyes for the purpose of increasing thesensitivity of a system that detects single particles are known in theart. Preferably, the modification improves the Stokes shift whilemaintaining a high quantum yield.

2 Quantum Dots

In some embodiments, the fluorescent label moiety that is used to detecta molecule in a sample using the analyzer systems of the invention is aquantum dot. Quantum dots (QDs), also known as semiconductornanocrystals or artificial atoms, are semiconductor crystals thatcontain anywhere between 100 to 1,000 electrons and range from 2-10 nm.Some QDs can be between 10-20 nm in diameter. QDs have high quantumyields, which makes them particularly useful for optical applications.QDs are fluorophores that fluoresce by forming excitons, which can bethought of the excited state of traditional fluorophores, but have muchlonger lifetimes of up to 200 nanoseconds. This property provides QDswith low photobleaching. The energy level of QDs can be controlled bychanging the size and shape of the QD, and the depth of the QDs'potential. One of the optical features of small excitonic QDs iscoloration, which is determined by the size of the dot. The larger thedot, the redder, or more towards the red end of the spectrum thefluorescence. The smaller the dot, the bluer or more towards the blueend it is. The bandgap energy that determines the energy and hence thecolor of the fluoresced light is inversely proportional to the square ofthe size of the QD. Larger QDs have more energy levels which are moreclosely spaced, thus allowing the QD to absorb photons containing lessenergy, i.e. those closer to the red end of the spectrum. Because theemission frequency of a dots dependent on the bandgap, it is thereforepossible to control the output wavelength of a dot with extremeprecision. In some embodiments the protein that is detected with thesingle particle analyzer system is labeled with a QD. In someembodiments, the single particle analyzer is used to detect a proteinlabeled with one QD and using a filter to allow for the detection ofdifferent proteins at different wavelengths.

QDs have broad excitation and narrow emission properties which when usedwith color filtering require only a single electromagnetic source formultiplex analysis of multiple targets in a single sample to resolveindividual signals. Thus, in some embodiments, the analyzer systemcomprises one continuous wave laser and particles that are each labeledwith one QD. Colloidally prepared QDs are free floating and can beattached to a variety of molecules via metal coordinating functionalgroups. These groups include but are not limited to thiol, amine,nitrile, phosphine, phosphine oxide, phosphoric acid, carboxylic acidsor other ligands By bonding appropriate molecules to the surface, thequantum dots can be dispersed or dissolved in nearly any solvent orincorporated into a variety of inorganic and organic films. Quantum dots(QDs) can be coupled to streptavidin directly through a maleimide estercoupling reaction or to antibodies through a meleimide-thiol couplingreaction. This yields a material with a biomolecule covalently attachedon the surface, which produces conjugates with high specific activity.In some embodiments, the protein that is detected with the singleparticle analyzer is labeled with one quantum dot. In some embodimentsthe quantum dot is between 10 and 20 nm in diameter. In otherembodiments, the quantum dot is between 2 and 10 nm in diameter. UsefulQuantum Dots include QD 605, QD 610, QD 655, and QD 705. A particularlypreferred Quantum Dot is QD 605.

C. Binding Partner-Fluorescent Moiety Compositions (Labels)

The labels of the invention generally contain a binding partner, e.g.,antibody, bound to a fluorescent moiety to provide the requisitefluorescence for detection and quantitation in the instruments describedherein. Any suitable combination of binding partner and fluorescentmoiety for detection in the single molecule detectors described hereinmay be used as a label in the invention. In some embodiments, theinvention provides a label for a cardiac troponin molecule, or fragment,complex, phosphorylated, or oxidized form thereof, where the labelincludes an antibody to a cardiac troponin and a fluorescent moiety. Theantibody may be any antibody as described above, e.g., an antibody tocTnT or cTnI. In some embodiments, the antibody is an antibody to cTnI.In some embodiments, the antibody is specific to a specific region ofthe cardiac troponin, e.g., specific to amino acids 27-41 of human cTnI.In some embodiments, the invention provides compositions comprising afluorescent moiety attached to an anti-cTnI antibody, e.g., a polyclonalantibody such as a goat polyclonal antibody from those designated G129Cavailable from BiosPacific, Emeryville. A fluorescent moiety may beattached such that the label is capable of emitting an average of atleast about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350,400, 500, 600, 700, 800, 900, or 1000, photons when simulated by a laseremitting light at the excitation wavelength of the moiety, where thelaser is focused on a spot of not less than about 5 microns in diameterthat contains the label, and wherein the total energy directed at thespot by the laser is no more than about 3 microJoules. In someembodiments, the fluorescent moiety may be a fluorescent moiety that iscapable of emitting an average of at least about 50, 100, 150, or 200photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the moiety, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules. The fluorescent moiety may be a fluorescentmoiety that includes one or more dye molecules with a structure thatincludes a substituted indolium ring system in which the substituent onthe 3-carbon of the indolium ring contains a chemically reactive groupor a conjugated substance group. The label composition may include afluorescent moiety that includes one or more dye molecules selected fromthe group consisting of ALEXA-FLUOR® 488, 532, 647, 700, or 750. Thelabel composition may include a fluorescent moiety that includes one ormore dye molecules selected from the group consisting of ALEXA-FLUOR®488, 532, 700, or 750. The label composition may include a fluorescentmoiety that includes one or more dye molecules that are ALEXA-FLUOR®488. The label composition may include a fluorescent moiety thatincludes one or more dye molecules that are ALEXA-FLUOR® 555. The labelcomposition may include a fluorescent moiety that includes one or moredye molecules that are ALEXA-FLUOR® 610. The label composition mayinclude a fluorescent moiety that includes one or more dye moleculesthat are ALEXA-FLUOR® 647. The label composition may include afluorescent moiety that includes one or more dye molecules that areALEXA-FLUOR® 680. The label composition may include a fluorescent moietythat includes one or more dye molecules that are ALEXA-FLUOR® 700. Thelabel composition may include a fluorescent moiety that includes one ormore dye molecules that are ALEXA-FLUOR® 750.

In some embodiments the invention provides a composition for thedetection of cardiac troponin I that includes an AlexFluor molecule,e.g. an ALEXA-FLUOR® molecule selected from the described groups, suchas an ALEXA-FLUOR® 647 molecule attached to a to an antibody, e.g., agoat polyclonal anti-cTnI antibody, specific for amino acids 27-41 ofhuman cTnI. In some embodiments the invention provides a composition forthe detection of cardiac troponin I that includes an average of 1 to 11,or about 2 to 10, or about 2 to 8, or about 2 to 6, or about 2 to 5, orabout 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 to 6, orabout 3 to 5, or about 4 to 10, or about 4 to 8, or about 4 to 6, orabout 2, 3, 4, 5, 6, or more than about 6 ALEXA-FLUOR® 647 moleculesmolecule attached an antibody, e.g., a goat polyclonal anti-cTnIantibody, specific for amino acids 27-41 of human cTnI. In someembodiments the invention provides a composition for the detection ofcardiac troponin I that includes an average of 1 to 11, or about 2 to10, or about 2 to 8, or about 2 to 6, or about 2 to 5, or about 2 to 4,or about 3 to 10, or about 3 to 8, or about 3 to 6, or about 3 to 5, orabout 4 to 10, or about 4 to 8, or about 4 to 6, or about 2, 3, 4, 5, 6,or more than about 6 ALEXA-FLUOR® 647 molecules molecule attached to anantibody, e.g., a goat polyclonal anti-cTnI antibody, specific for aminoacids 27-41 of human cTnI. In some embodiments the invention provides acomposition for the detection of cardiac troponin I that includes anaverage of about 2 to 10 ALEXA-FLUOR® 647 molecules molecule attached toan antibody, e.g., a goat polyclonal anti-cTnI antibody, specific foramino acids 27-41 of human cTnI. In some embodiments the inventionprovides a composition for the detection of cardiac troponin I thatincludes an average of about 2 to 8 ALEXA-FLUOR® 647 molecules moleculeattached to an antibody, e.g., a goat polyclonal anti-cTnI antibody,specific for amino acids 27-41 of human cTnI. In some embodiments theinvention provides a composition for the detection of cardiac troponin Ithat includes an average of about 2 to 6 ALEXA-FLUOR® 647 moleculesmolecule attached to an antibody, e.g., a goat polyclonal anti-cTnIantibody, specific for amino acids 27-41 of human cTnI. In someembodiments the invention provides a composition for the detection ofcardiac troponin I that includes an average of about 2 to 4 ALEXA-FLUOR®647 molecules molecule attached to an antibody, e.g., a goat polyclonalanti-cTnI antibody, specific for amino acids 27-41 of human cTnI. Insome embodiments the invention provides a composition for the detectionof cardiac troponin I that includes an average of about 3 to 8ALEXA-FLUOR® 647 molecules molecule attached to an antibody, e.g., agoat polyclonal anti-cTnI antibody, specific for amino acids 27-41 ofhuman cTnI. In some embodiments the invention provides a composition forthe detection of cardiac troponin I that includes an average of about 3to 6 ALEXA-FLUOR® 647 molecules molecule attached to an antibody, e.g.,a goat polyclonal anti-cTnI antibody, specific for amino acids 27-41 ofhuman cTnI. In some embodiments the invention provides a composition forthe detection of cardiac troponin I that includes an average of about 4to 8 ALEXA-FLUOR® 647 molecules molecule attached to an antibody, e.g.,a goat polyclonal anti-cTnI antibody, specific for amino acids 27-41 ofhuman cTnI.

Attachment of the fluorescent moiety, or fluorescent entities that makeup the fluorescent moiety, to the binding partner, e.g., antibody, maybe by any suitable means; such methods are well-known in the art andexemplary methods are given in the Examples. In some embodiments, afterattachment of the fluorescent moiety to the binding partner to form alabel for use in the methods of the invention, and prior to the use ofthe label for labeling the protein of interest, it is useful to performa filtration step. E.g., an antibody-dye label may be filtered prior touse, e.g., through a 0.2 micron filter, or any suitable filter forremoving aggregates. Other reagents for use in the assays of theinvention may also be filtered, e.g., e.g., through a 0.2 micron filter,or any suitable filter. Without being bound by theory, it is thoughtthat such filtration removes a portion of the aggregates of the, e.g.,antibody-dye labels. As such aggregates will bind as a unit to theprotein of interest, but upon release in elution buffer are likely todisaggregate, false positives may result; i.e., several labels will bedetected from an aggregate that has bound to only a single proteinmolecule of interest. Regardless of theory, filtration has been found toreduce false positives in the subsequent assay and to improve accuracyand precision.

IV. Highly Sensitive Analysis of Cardiac Troponin

In one aspect, the invention provides a method for determining thepresence or absence of a single molecule of cardiac troponin or afragment or complex thereof in a sample, by i) labeling the molecule,fragment, or complex, if present, with a label; and ii) detecting thepresence or absence of the label, where the detection of the presence ofthe label indicates the presence of the single molecule, fragment, orcomplex of cardiac troponin in the sample. As used herein, “molecule ofcardiac troponin” includes a molecule that contains substantially theentire naturally-occurring amino acid sequence of the particular type ofcardiac troponin, including post-translationally modified forms, e.g.,phosphorylated forms, as well as oxidized or otherwise chemicallyaltered forms. As used herein, a “fragment” of a molecule includes amolecule of cardiac troponin that contains less than the entirenaturally-occurring amino acid sequence, including modifications as forthe entire molecule. As used herein, a “complex” of a molecule ofcardiac troponin includes a molecule of cardiac troponin or a fragmentthat is associated with one or more other molecules or substances, e.g.,that is associated with one or more other molecules of cardiac troponin.In some embodiments, the method is capable of detecting the troponin ata limit of detection of less than about 100, 80, 60, 50, 40, 30, 20, 15,12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3,0.2. or 0.1 pg/ml. In some embodiments, the method is capable ofdetecting the troponin at a limit of detection of less than about 100pg/ml. In some embodiments, the method is capable of detecting thetroponin at a limit of detection of less than about 50 pg/ml. In someembodiments, the method is capable of detecting the troponin at a limitof detection of less than about 20 pg/ml. In some embodiments, themethod is capable of detecting the troponin at a limit of detection ofless than about 10 pg/ml. In some embodiments, the method is capable ofdetecting the troponin at a limit of detection of less than about 5pg/ml. In some embodiments, the method is capable of detecting thetroponin at a limit of detection of less than about 3 pg/ml. In someembodiments, the method is capable of detecting the troponin at a limitof detection of less than about 1 pg/ml. Detection limits may bedetermined by use of the appropriate National Institute of Standards andTechnology reference standard material, e.g., standard cTnI.

The methods also provide methods of determining a concentration ofcardiac troponin in a sample by detecting single molecules of troponinin the sample. The “detecting” of a single molecule of troponin includesdetecting the molecule directly or indirectly. In the case of indirectdetection, labels that corresponds to single molecules of cardiactroponin, e.g., a labels that have been attached to the single moleculesof cardiac troponin, may be detected.

Types of cardiac troponin for detection are as described herein, e.g.,cTnT, cTnI, total cardiac troponin (e.g., total cTnI or total cTnT) orfree, complexed, or fragments of cardiac troponin. In some embodiments,total cardiac troponin is detected and/or quantitated. In someembodiments, total cTnT is detected. In some embodiments, total cTnI isdetected and/or quantitated.

The methods also provide for the measurement of low levels of biomarkersother than troponin. For example, the detection of cytokines in plasmain the absence of inflammation, at, for example, the level of cytokinesknown to exist in the circulating plasma proteome of a healthyindividual (ref. 2). Another non-limiting example is the detection andmeasurement of p24, a viral protein present in HIV infected individuals,which is present in the early stages of HIV infection well before HIVantibodies are produced and can be detected (ref. 12).

A. Sample

The sample may be any suitable sample Typically, the sample is abiological sample, e.g., a biological fluid. Such fluids include,without limitation, exhaled breath condensate (EBC), bronchoalveolarlavage fluid (BAL), blood, serum, plasma, urine, cerebrospinal fluid,pleural fluid, synovial fluid, peritoneal fluid, amniotic fluid, gastricfluid, lymph fluid, interstitial fluid, tissue homogenate, cellextracts, saliva, sputum, stool, physiological secretions, tears, mucus,sweat, milk, semen, seminal fluid, vaginal secretions, fluid from ulcersand other surface eruptions, blisters, and abscesses, and extracts oftissues including biopsies of normal, malignant, and suspect tissues orany other constituents of the body which may contain the target particleof interest. Other similar specimens such as cell or tissue culture orculture broth are also of interest.

In some embodiments, the sample is a blood sample. In some embodimentsthe sample is a plasma sample. In some embodiments the sample is a serumsample

B. Sample Preparation

In general, any method of sample preparation may be used that produces alabel corresponding to a molecule of cardiac troponin that is wished tobe measured, where the label is detectable in the instruments describedherein. As is known in the art, sample preparation in which a label isadded to one or more particles may be performed in a homogeneous orheterogeneous format. In some embodiments, the sample preparation isformed in a homogenous format. In analyzer system employing a homogenousformat, unbound label is not removed from the sample See, e.g., U.S.patent application Ser. No. 11/048,660, incorporated by reference hereinin its entirety. In some embodiments, the particle or particles ofinterest are labeled by addition of labeled antibody or antibodies thatbind to the particle or particles of interest.

In some embodiments, a heterogeneous assay format is used, where,typically, a step is employed for removing unbound label. Such assayformats are well-known in the art. One particularly useful assay formatis a sandwich assay, e.g., a sandwich immunoassay. In this format, themolecule of interest, e.g., marker of a biological state, is captured,e.g., on a solid support, using a capture binding partner. Unwantedmolecules and other substances may then optionally be washed away,followed by binding of a label comprising a detection binding partnerand a detectable label, e.g., fluorescent moiety. Further washes removeunbound label, then the detectable label is released, usually though notnecessarily still attached to the detection binding partner. Inalternative embodiments, sample and label are added to the capturebinding partner without a wash in between, e.g., at the same time. Othervariations will be apparent to one of skill in the art.

In some embodiments, the method for detecting troponin particles uses asandwich assay with antibodies, e.g., monoclonal antibodies as capturebinding partners. The method comprises binding troponin molecules in asample to a capture antibody that is immobilized on a binding surface,and binding the detection antibody to the troponin molecule to form a“sandwich” complex. The detection antibody comprises a detectablefluorescent label, as described herein, which is detected, e.g., usingthe single molecule analyzers of the invention. Both the capture anddetection antibodies specifically bind troponin. Many example ofsandwich immunoassays are known, and some are described in U.S. Pat. No.4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al.,both of which are incorporated herein by reference. Further examplesspecific to cardiac troponin are described in the Examples.

The capture binding partner may be attached to a solid support, e.g., amicrotiter plate or paramagnetic beads. In some embodiments, theinvention provides a binding partner for a cardiac troponin attached toa paramagnetic bead. Any suitable binding partner that is specific forthe type of cardiac troponin that it is wished to capture may be used.The binding partner may be an antibody, e.g., a monoclonal antibody. Theantibody may be specific for free cardiac troponin (cTnI or cTnT) or forcomplexed cardiac troponin, modified cardiac troponin, or fragments ofcardiac troponin, as described herein, or specific to all orsubstantially all forms of cardiac troponin, e.g., cTnI or cTnT, likelyto be found in the sample of interest. Production and sources ofantibodies to cardiac troponin are described elsewhere herein. Preferredantibodies for measuring total troponin are those that not substantiallyaffected by heparin, phosphorylation, oxidation and troponin complexformation, and that do not cross-react with skeletal muscle troponin,e.g., troponin I. In some embodiments, the antibody is specific for aspecific region of a cardiac troponin. In some embodiments, the regionincludes amino acids 41-49 of human cardiac troponin I. In someembodiments, the region includes amino acids 87-91 of human cardiactroponin I. Such antibodies are well-known in the art and are availablefrom, e.g. BiosPacific, Emeryville, Calif. An example of a captureantibody useful in embodiments of the invention is an antibody, e.g., amonoclonal antibody, that reacts with free cardiac troponin I (cTnI)a.a. 41-49 and cTnI forming complexes with other troponin components.Preferably, this antibody is not affected by heparin, phosphorylation,oxidation and troponin complex formation, and does not cross-react withskeletal muscle troponin I. An exemplary antibody of this type isMonoclonal Antibody Clone Number A34650228P, available from BiosPacific,Emeryville, Calif. Another example of a capture antibody useful inembodiments of the invention is an antibody, e.g., a monoclonalantibody, that reacts with free cardiac troponin I (cTnI) a.a. 87-91 andcTnI forming complexes with other troponin components. Preferably, thisantibody is not affected by heparin, phosphorylation, oxidation andtroponin complex formation, and does not cross-react with skeletalmuscle troponin I. An exemplary antibody of this type is MonoclonalAntibody Clone Number A34440228P, available from BiosPacific,Everyville, Calif. It will be appreciated that antibodies identifiedherein as useful as a capture antibody may also be useful as detectionantibodies, and vice versa.

The attachment of the binding partner, e.g., antibody, to the solidsupport may be covalent or noncovalent. In some embodiments, theattachment is noncovalent. An example of a noncovalent attachmentwell-known in the art is biotin-avidin/streptavidin interactions. Thus,in some embodiments, a solid support, e.g., a microtiter plate or aparamagnetic bead, is attached to the capture binding partner, e.g.,antibody, through noncovalent attachment, e.g.,biotin-avidin/streptavidin interactions. In some embodiments, theattachment is covalent. Thus, in some embodiments, a solid support,e.g., a microtiter plate or a paramagnetic bead, is attached to thecapture binding partner, e.g., antibody, through covalent attachment.Covalent attachment in which the orientation of the capture antibody issuch that capture of the molecule of interest is optimized is especiallyuseful. For example, in some embodiments a solid support, e.g., amicrotiter plate or a paramagnetic microparticle, may be used in whichthe attachment of the binding partner, e.g., antibody, is an orientedattachment, e.g., a covalent oriented attachment.

An exemplary protocol for oriented attachment of an antibody to a solidsupport is as follows: IgG is dissolved in 0.1M sodium acetate buffer,pH 5.5 to a final concentration of 1 mg/ml. An equal volume of ice-cold20 mM sodium periodate in 0.1 M sodium acetate, pH 5.5 is added. The IgGis allowed to oxidize for ½ hour on ice. Excess periodate reagent isquenched by the addition of 0.15 volume of 1 M glycerol. Low molecularweight byproducts of the oxidation reaction are removed byultrafiltration. The oxidized IgG fraction is diluted to a suitableconcentration (typically 0.5 micrograms IgG per ml) and reacted withhydrazide-activated multiwell plates for at least two hours at roomtemperature. Unbound IgG is removed by washing the multiwell plate withborate buffered saline or another suitable buffer. The plate may bedried for storage, if desired. A similar protocol may be followed formicrobeads if the material of the microbead is suitable for suchattachment.

In some embodiments, the solid support is a microtiter plate. In someembodiments, the solid support is a paramagnetic bead. An exemplaryparamagnetic bead is Streptavidin C1(Dynal, 650.01-03). Other suitablebeads will be apparent to those of skill in the art. Methods forattachment of antibodies to paramagnetic beads are well-known in theart. One example is given in the Examples.

The cardiac troponin of interest is contacted with the capture bindingpartner, e.g., capture antibody immobilized on a solid support. Somesample preparation may be used; e.g., preparation of serum from bloodsamples or concentration procedures before the sample is contacted withthe capture antibody. Protocols for binding of proteins in immunoassaysare well-known in the art and are included in the Examples.

The time allowed for binding will vary depending on the conditions; itwill be apparent that shorter binding times are desirable in somesettings, especially in a clinical setting. The use of, e.g.,paramagnetic beads can reduce the time required for binding. In someembodiments, the time allowed for binding of the protein of interest tothe capture binding partner, e.g., antibody, is less that about 12, 10,8, 6, 4, 3, 2, or 1 hours, or less than about 60, 50, 40, 30, 25, 20,15, 10, or 5 minutes. In some embodiments, the time allowed for bindingof the protein of interest to the capture binding partner, e.g.,antibody, is less than about 60 minutes. In some embodiments, the timeallowed for binding of the protein of interest to the capture bindingpartner, e.g., antibody, is less that about 40 minutes. In someembodiments, the time allowed for binding of the protein of interest tothe capture binding partner, e.g., antibody, is less that about 30minutes. In some embodiments, the time allowed for binding of theprotein of interest to the capture binding partner, e.g., antibody, isless that about 20 minutes. In some embodiments, the time allowed forbinding of the protein of interest to the capture binding partner, e.g.,antibody, is less that about 15 minutes. In some embodiments, the timeallowed for binding of the protein of interest to the capture bindingpartner, e.g., antibody, is less that about 10 minutes. In someembodiments, the time allowed for binding of the protein of interest tothe capture binding partner, e.g., antibody, is less that about 5minutes.

In some embodiments, following the binding of the troponin particles tothe capture binding partner, e.g., capture antibody, particles that mayhave bound nonspecifically, as well as other unwanted substances in thesample, are washed away leaving substantially only specifically boundtroponin particles. In other embodiments, no wash is used betweenadditions of sample and label; it will be appreciated that this reducessample preparation time even further. Thus, in some embodiments, thetime allowed for both binding of the protein of interest to the capturebinding partner, e.g., antibody, and binding of the label to the proteinof interest, is less that about 12, 10, 8, 6, 4, 3, 2, or 1 hours, orless than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In someembodiments, the time allowed for both binding of the protein ofinterest to the capture binding partner, e.g., antibody, and binding ofthe label to the protein of interest, is less that about 60 minutes. Insome embodiments, the time allowed for both binding of the protein ofinterest to the capture binding partner, e.g., antibody, and binding ofthe label to the protein of interest, is less than about 40 minutes. Insome embodiments, the time allowed for both binding of the protein ofinterest to the capture binding partner, e.g., antibody, and binding ofthe label to the protein of interest, is less than about 30 minutes. Insome embodiments, the time allowed for both binding of the protein ofinterest to the capture binding partner, e.g., antibody, and binding ofthe label to the protein of interest, is less than about 20 minutes. Insome embodiments, the time allowed for both binding of the protein ofinterest to the capture binding partner, e.g., antibody, and binding ofthe label to the protein of interest, is less than about 15 minutes. Insome embodiments, the time allowed for both binding of the protein ofinterest to the capture binding partner, e.g., antibody, and binding ofthe label to the protein of interest, is less than about 10 minutes. Insome embodiments, the time allowed for both binding of the protein ofinterest to the capture binding partner, e.g., antibody, and binding ofthe label to the protein of interest, is less than about 5 minutes.

Some immunoassay diagnostic reagents including the capture and signalantibodies used to measure the target analytes may be derived from thesera of animals Endogenous human heterophilic antibodies, or humananti-animal antibodies, which have the ability to bind toimmunoglobulins of other species, are present in the serum or plasma ofmore than 10% of patients. These circulating heterophile antibodies mayinterfere with immunoassay measurements. In sandwich immunoassays, theseheterophilic antibodies can either bridge the capture and detection(diagnostic) antibodies, thereby producing a false-positive signal, orthey may block the binding of the diagnostic antibodies, therebyproducing a false-negative signal. In competitive immunoassays, theheterophile antibodies may bind to the analytic antibody and inhibit itsbinding to the troponin. They also may either block or augment theseparation of the antibody-troponin complex from free troponin,especially when antispecies antibodies are used in the separationsystems. Therefore, the impact of these heterophile antibodyinterferences are difficult to predict. Thus, it would be advantageousto block the binding of any heterophilic antibodies. In some embodimentsof the invention, the immunoassay includes the step of depleting thesample of heterophile antibodies using one or more heterophile antibodyblockers. Methods for removing heterophile antibodies from samples thatare to be tested in immunoassays are known and include: heating thespecimen in a sodium acetate buffer, pH 5.0, for 15 minutes at 90° C.and centrifuging at 1200 g for 10 minutes, or the heterophile antibodiescan be precipitated using polyethylene glycol (PEG); immunoextractingthe interfering heterophile immunoglobulins from the specimen usingprotein A or protein G; or adding nonimmune mouse IgG. Embodiments ofthe methods of the invention contemplate preparing the sample prior toanalysis with the single molecule detector. The appropriateness of themethod of pretreatment may be determined. Biochemicals to minimizeimmunoassay interference caused by heterophile antibodies arecommercially available. For example, a product called MAK33, which is anIgG1 monoclonal antibody to h-CK-MM, may be obtained from BoehringerMannheim. The MAK33 plus product contains a combination of IgG1 andIgG1-Fab. The polyMAK33 contains IgG1-Fab polymerized with IgG1, and thepolyMAC 2b/2a contains IgG2a-Fab polymerized with IgG2b. A secondcommercial source of biochemicals to neutralize heterophile antibodiesis Immunoglobulin Inhibiting Reagent marketed by Bioreclamation Inc,East Meadow, N.Y. This product is a preparation of immunoglobulins (IgGand IgM) from multiple species, mainly murine IgG2a, IgG2b, and IgG3from Balb/c mice. In some embodiments the heterophile antibody may beimmunoextracted from the sample using methods known in the art e.g.depleting the sample of the heterophile antibody by binding theinterfering antibody to protein A or G. In some embodiments, theheterophile antibody is neutralized using one or more heterophileantibody blockers. Heterophile blockers may be selected from the groupconsisting of anti-isotype heterophile antibody blockers, anti-idiotypeheterophile antibody blockers, and anti-anti-idiotype heterophileantibody blockers. In some embodiments a combination of heterophileantibody blockers may be used.

Label is added either with or following the addition of sample andwashing. Protocols for binding of antibody and other immunolabels toproteins and other molecules are well-known in the art. If the labelbinding step is separate from capture binding, the time allowed forlabel binding can be important, e.g., in the clinical setting. In someembodiments, the time allowed for binding of the protein of interest tothe label, e.g., antibody-dye, is less than about 12, 10, 8, 6, 4, 3, 2,or 1 hours, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5minutes. In some embodiments, the time allowed for binding of theprotein of interest to the label, e.g., antibody-dye, is less than about60 minutes. In some embodiments, the time allowed for binding of theprotein of interest to the label, e.g., antibody-dye, is less than about40 minutes. In some embodiments, the time allowed for binding of theprotein of interest to the label, e.g., antibody-dye, is less than about30 minutes. In some embodiments, the time allowed for binding of theprotein of interest to the label, e.g., antibody-dye, is less than about20 minutes. In some embodiments, the time allowed for binding of theprotein of interest to the label, e.g., antibody-dye, is less than about15 minutes. In some embodiments, the time allowed for binding of theprotein of interest to the label, e.g., antibody-dye, is less than about10 minutes. In some embodiments, the time allowed for binding of theprotein of interest to the label, e.g., antibody-dye, is less than about5 minutes. Excess label is removed by washing.

Label is then eluted from the protein of interest. Preferred elutionbuffers are effective in releasing the label without generatingsignificant background. It is also useful if the elution buffer isbacteriostatic. Elution buffers of use in the invention include achaotrope, e.g., urea or a guanidinium compound; a buffer, e.g., boratebuffered saline; a protein carrier, e.g., an albumin, such as human,bovine, or fish albumin, or an IgG, to coat the wall of the capillarytube in the detection instrument; and a surfactant, e.g., an ionic ornonionic detergent, selected so as to produce a relatively lowbackground, e.g., Tween 20, Triton X-100, or SDS.

The elution buffer/label aliquot that is sampled into the singlemolecule detector is referred to as the “processing sample,” todistinguish it from the original sample which was obtained from anindividual.

In another embodiment, the solid phase binding assay may employ acompetitive binding assay format. One such method comprises a)competitively binding to a capture antibody immobilized on a bindingsurface i) a troponin particle in a sample and ii) a labeled analog ofthe troponin particle comprising a detectable label (the detectionreagent) and b) measuring the amount of the label using a singleparticle analyzer. Another such method comprises a) competitivelybinding to an antibody having a detectable label (the detection reagent)i) a troponin particle in a sample and ii) an analog of troponinparticle that is immobilized on a binding surface (the capture reagent)and b) measuring the amount of the label using a single particleanalyzer. An “analog of a troponin” refers, herein, to a species thatcompetes with troponin for binding to a capture antibody. Examples ofcompetitive immunoassays are disclosed in U.S. Pat. No. 4,235,601 toDeutsch et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No.5,208,535 to Buechler et al., all of which are incorporated herein byreference.

C. Detection of Troponin and Determination of Concentration

Following elution, the label is run through a single molecule detectorin e.g., the elution buffer. A processing sample may contain no label, asingle label, or a plurality of labels. The number of labels correspondsor is proportional to (if dilutions or fractions of samples are used)the number of molecules of cardiac troponin captured during the capturestep.

Any suitable single molecule detector capable of detecting the labelused with the protein of interest may be used. Suitable single moleculedetectors are described herein. Typically the detector will be part of asystem that includes an automatic sampler for sampling prepared samples,and, optionally, a recovery system to recover samples.

In some embodiments, the processing sample is analyzed in a singlemolecule analyzer that utilizes a capillary flow system, and thatincludes a capillary flow cell, a laser to illuminate an interrogationspace in the capillary through which processing sample is passed, adetector to detect radiation emitted from the interrogation space, and asource of motive force to move a processing sample through theinterrogation space. In some embodiments, the single molecule analyzerfurther comprises a microscope objective lens that collects lightemitted from processing sample as it passes through the interrogationspace, e.g., a high numerical aperture microscope objective. In someembodiments, the laser and detector are in a confocal arrangement. Insome embodiments, the laser is a continuous wave laser. In someembodiments, the detector is a avalanche photodiode detector. In someembodiments, the source of motive force is a pump to provide pressure.In some embodiments, the invention provides an analyzer system thatincludes a sampling system capable of automatically sampling a pluralityof samples providing a fluid communication between a sample containerand the interrogation space. In some embodiments, the interrogationspace has a volume of between about 0.001 and 500 pL, or between about0.01 pL and 100 pL, or between about 0.01 pL and 10 pL, or between about0.01 pL and 5 pL, or between about 0.01 pL and 0.5 pL, or between about0.02 pL and about 300 pL, or between about 0.02 pL and about 50 pL orbetween about 0.02 pL and about 5 pL or between about 0.02 pL and about0.5 pL or between about 0.02 pL and about 2 pL, or between about 0.05 pLand about 50 pL, or between about 0.05 pL and about 5 pL, or betweenabout 0.05 pL and about 0.5 pL, or between about 0.05 pL and about 0.2pL, or between about 0.1 pL and about 25 pL. In some embodiments, theinterrogation space has a volume between about 0.004 pL and 100 pL. Insome embodiments, the interrogation space has a volume between about0.02 pL and 50 pL. In some embodiments, the interrogation space has avolume between about 0.001 pL and 10 pL. In some embodiments, theinterrogation space has a volume between about 0.001 pL and 10 pL. Insome embodiments, the interrogation space has a volume between about0.01 pL and 5 pL. In some embodiments, the interrogation space has avolume between about 0.02 pL and about 5 pL. In some embodiments, theinterrogation space has a volume between about 0.05 pL and 5 pL In someembodiments, the interrogation space has a volume between about 0.05 pLand 10 pL. In some embodiments, the interrogation space has a volumebetween about 0.5 pL and about 5 pL. In some embodiments, theinterrogation space has a volume between about 0.02 pL and about 0.5 pL.

In some embodiments, the single molecule detector used in the methods ofthe invention utilizes a capillary flow system, and includes a capillaryflow cell, a continuous wave laser to illuminate an interrogation spacein the capillary through which processing sample is passed, a highnumerical aperture microscope objective lens that collects light emittedfrom processing sample as it passes through the interrogation space, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a pump to provide pressure to move a processingsample through the interrogation space, where the interrogation space isbetween about 0.02 pL and about 50 pL. In some embodiments, the singlemolecule detector used in the methods of the invention utilizes acapillary flow system, and includes a capillary flow cell, a continuouswave laser to illuminate an interrogation space in the capillary throughwhich processing sample is passed, a high numerical aperture microscopeobjective lens that collects light emitted from processing sample as itpasses through the interrogation space wherein the lens has a numericalaperture of at least about 0.8, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a pump toprovide pressure to move a processing sample through the interrogationspace, where the interrogation space is between about 0.004 pL and about100 pL. In some embodiments, the single molecule detector used in themethods of the invention utilizes a capillary flow system, and includesa capillary flow cell, a continuous wave laser to illuminate aninterrogation space in the capillary through which processing sample ispassed, a high numerical aperture microscope objective lens thatcollects light emitted from processing sample as it passes through theinterrogation space wherein the lens has a numerical aperture of atleast about 0.8, an avalanche photodiode detector to detect radiationemitted from the interrogation space, and a pump to provide pressure tomove a processing sample through the interrogation space, where theinterrogation space is between about 0.05 pL and about 10 pL. In someembodiments, the single molecule detector used in the methods of theinvention utilizes a capillary flow system, and includes a capillaryflow cell, a continuous wave laser to illuminate an interrogation spacein the capillary through which processing sample is passed, a highnumerical aperture microscope objective lens that collects light emittedfrom processing sample as it passes through the interrogation spacewherein the lens has a numerical aperture of at least about 0.8, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a pump to provide pressure to move a processingsample through the interrogation space, where the interrogation space isbetween about 0.05 pL and about 5 pL. In some embodiments, the singlemolecule detector used in the methods of the invention utilizes acapillary flow system, and includes a capillary flow cell, a continuouswave laser to illuminate an interrogation space in the capillary throughwhich processing sample is passed, a high numerical aperture microscopeobjective lens that collects light emitted from processing sample as itpasses through the interrogation space wherein the lens has a numericalaperture of at least about 0.8, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a pump toprovide pressure to move a processing sample through the interrogationspace, where the interrogation space is between about 0.5 pL and about 5pL.

In some embodiments, the single molecule detector is capable ofdetermining a concentration for a molecule of interest in a sample wheresample may range in concentration over a range of at least about100-fold, or 1000-fold, or 10,000-fold, or 100,000-fold, or 300,00-fold,or 1,000,000-fold, or 10,000,000-fold, or 30,000,000-fold.

In some embodiments, the methods of the invention utilize a singlemolecule detector capable detecting a difference of less than about 50%,40%, 30%, 20%, 15%, or 10% in concentration of an analyte between afirst sample and a second sample that are introduced into the detector,where the volume of the first sample and said second sample introducedinto the analyzer is less than about 100, 90, 80, 70, 60, 50, 40, 30,20, 15, 10, 5, 4, 3, 2, or 1 ul, and wherein the analyte is present at aconcentration of less than about 100, 90, 80, 70, 60, 50, 40, 30, 20,15, 10, 5, 4, 3, 2, or 1 femtomolar. In some embodiments, the methods ofthe invention utilize a single molecule detector capable detecting adifference of less than about 50% in concentration of an analyte betweena first sample and a second sample that are introduced into thedetector, where the volume of the first sample and said second sampleintroduced into the analyzer is less than about 100 ul, and wherein theanalyte is present at a concentration of less than about 100 femtomolar.In some embodiments, the methods of the invention utilize a singlemolecule detector capable detecting a difference of less than about 40%in concentration of an analyte between a first sample and a secondsample that are introduced into the detector, where the volume of thefirst sample and said second sample introduced into the analyzer is lessthan about 50 ul, and wherein the analyte is present at a concentrationof less than about 50 femtomolar. In some embodiments, the methods ofthe invention utilize a single molecule detector capable detecting adifference of less than about 20% in concentration of an analyte betweena first sample and a second sample that are introduced into thedetector, where the volume of the first sample and said second sampleintroduced into the analyzer is less than about 20 ul, and wherein theanalyte is present at a concentration of less than about 20 femtomolar.In some embodiments, the methods of the invention utilize a singlemolecule detector capable detecting a difference of less than about 20%in concentration of an analyte between a first sample and a secondsample that are introduced into the detector, where the volume of thefirst sample and said second sample introduced into the analyzer is lessthan about 10 ul, and wherein the analyte is present at a concentrationof less than about 10 femtomolar. In some embodiments, the methods ofthe invention utilize a single molecule detector capable detecting adifference of less than about 20% in concentration of an analyte betweena first sample and a second sample that are introduced into thedetector, where the volume of the first sample and said second sampleintroduced into the analyzer is less than about 5 ul, and wherein theanalyte is present at a concentration of less than about 5 femtomolar.

The single molecule detector and systems are described in more detailbelow. Further embodiments of single molecule analyzers useful in themethods of the invention, such as detectors with more than oneinterrogation window, detectors utilize electrokinetic orelectrophoretic flow, and the like, may be found in U.S. patentapplication Ser. No. 11/048,660, incorporated by reference herein in itsentirety.

Between runs the instrument may be washed. A wash buffer that maintainsthe salt and surfactant concentrations of the sample may be used in someembodiments to maintain the conditioning of the capillary; i.e., to keepthe capillary surface relatively constant between samples to reducevariability.

A feature that contributes to the extremely high sensitivity of theinstruments and methods of the invention is the method of detecting andcounting labels, which, in some embodiments, are attached to singlemolecules to be detected or, more typically, correspond to a singlemolecule to be detected. Briefly, the processing sample flowing throughthe capillary is effectively divided into a series of detection events,by subjecting a given interrogation space of the capillary to EMradiation from a laser that emits light at an appropriate excitationwavelength for the fluorescent moiety used in the label for apredetermined period of time, and detecting photons emitted during thattime. Each predetermined period of time is a “bin.” If the total numberof photons detected in a given bin exceeds a predetermined thresholdlevel, a detection event is registered for that bin, i.e., a label hasbeen detected. If the total number of photons is not at thepredetermined threshold level, no detection event is registered. In someembodiments, processing sample concentration is dilute enough that, fora large percentage of detection events, the detection event representsonly one label passing through the window, which corresponds to a singlemolecule of interest in the original sample, that is, few detectionevents represent more than one label in a single bin. In someembodiments, further refinements are applied to allow greaterconcentrations of label in the processing sample to be detectedaccurately, i.e., concentrations at which the probability of two or morelabels being detected as a single detection event is no longerinsignificant.

Although other bin times may be used without departing from the scope ofthe present invention, in some embodiments the bin times are selected inthe range of about 1 microsecond to about 5 ms. In some embodiments, thebin time is more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750,800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. In someembodiments, the bin time is less than about 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600,700, 750, 800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. Insome embodiments, the bin time is about 1 to 1000 microseconds. In someembodiments, the bin time is about 1 to 750 microseconds. In someembodiments, the bin time is about 1 to 500 microseconds. In someembodiments, the bin time is about 1 to 250 microseconds. In someembodiments, the bin time is about 1 to 100 microseconds. In someembodiments, the bin time is about 1 to 50 microseconds. In someembodiments, the bin time is about 1 to 40 microseconds. In someembodiments, the bin time is about 1 to 30 microseconds. In someembodiments, the bin time is about 1 to 500 microseconds. In someembodiments, the bin time is about 1 to 20 microseconds. In someembodiments, the bin time is about 1 to 10 microseconds. In someembodiments, the bin time is about 1 to 500 microseconds. In someembodiments, the bin time is about 1 to 5 microseconds. In someembodiments, the bin time is about 5 to 500 microseconds. In someembodiments, the bin time is about 5 to 250 microseconds. In someembodiments, the bin time is about 5 to 100 microseconds. In someembodiments, the bin time is about 5 to 50 microseconds. In someembodiments, the bin time is about 5 to 20 microseconds. In someembodiments, the bin time is about 5 to 10 microseconds. In someembodiments, the bin time is about 10 to 500 microseconds. In someembodiments, the bin time is about 10 to 250 microseconds. In someembodiments, the bin time is about 10 to 100 microseconds. In someembodiments, the bin time is about 10 to 50 microseconds. In someembodiments, the bin time is about 10 to 30 microseconds. In someembodiments, the bin time is about 10 to 20 microseconds. In someembodiments, the bin time is about 5 microseconds. In some embodiments,the bin time is about 5 microseconds. In some embodiments, the bin timeis about 6 microseconds. In some embodiments, the bin time is about 7microseconds. In some embodiments, the bin time is about 8 microseconds.In some embodiments, the bin time is about 9 microseconds. In someembodiments, the bin time is about 10 microseconds. In some embodiments,the bin time is about 11 microseconds. In some embodiments, the bin timeis about 12 microseconds. In some embodiments, the bin time is about 13microseconds. In some embodiments, the bin time is about 14microseconds. In some embodiments, the bin time is about 5 microseconds.In some embodiments, the bin time is about 15 microseconds. In someembodiments, the bin time is about 16 microseconds. In some embodiments,the bin time is about 17 microseconds. In some embodiments, the bin timeis about 18 microseconds. In some embodiments, the bin time is about 19microseconds. In some embodiments, the bin time is about 20microseconds. In some embodiments, the bin time is about 25microseconds. In some embodiments, the bin time is about 30microseconds. In some embodiments, the bin time is about 40microseconds. In some embodiments, the bin time is about 50microseconds. In some embodiments, the bin time is about 100microseconds. In some embodiments, the bin time is about 250microseconds. In some embodiments, the bin time is about 500microseconds. In some embodiments, the bin time is about 750microseconds. In some embodiments, the bin time is about 1000microseconds.

In some embodiments, the background noise level is determined from themean noise level, or the root-mean-square noise. In other cases, atypical noise value or a statistical value is chosen. In most cases, thenoise is expected to follow a Poisson distribution. Thus, in someembodiments, determining the concentration of a particle-label complexin a sample comprises determining the background noise level.

Thus, as a label flows through the capillary flow cell, it is irradiatedby the laser beam to generate a burst of photons. The photons emitted bythe label are discriminated from background light or background noiseemission by considering only the bursts of photons that have energyabove a predetermined threshold energy level which accounts for theamount of background noise that is present in the sample Backgroundnoise typically comprises low frequency emission produced, for example,by the intrinsic fluorescence of non-labeled particles that are presentin the sample, the buffer or diluent used in preparing the sample foranalysis, Raman scattering and electronic noise. In some embodiments,the value assigned to the background noise is calculated as the averagebackground signal noise detected in a plurality of bins, which aremeasurements of photon signals that are detected in an interrogationspace during a predetermined length of time. Thus in some embodiments,background noise is calculated for each sample as a number specific tothat sample

Given the value for the background noise, the threshold energy level canbe assigned. As discussed above, the threshold value is determined todiscriminate true signals (due to fluorescence of a label) from thebackground noise. Care must be taken in choosing a threshold value suchthat the number of false positive signals from random noise is minimizedwhile the number of true signals which are rejected is also minimizedMethods for choosing a threshold value include determining a fixed valueabove the noise level and calculating a threshold value based on thedistribution of the noise signal. In one embodiment, the threshold isset at a fixed number of standard deviations above the background level.Assuming a Poisson distribution of the noise, using this method one canestimate the number of false positive signals over the time course ofthe experiment. In some embodiments, the threshold level is calculatedas a value of 4 sigma above the background noise. For example, given anaverage background noise level of 200 photons, the analyzer systemestablishes a threshold level of 4√200 above the averagebackground/noise level of 200 photons to be 256 photons. Thus, in someembodiments, determining the concentration of a label in a sampleincludes establishing the threshold level above which photon signalsrepresent the presence of a label. Conversely, photon signals that havean energy level that is not greater than that of the threshold levelindicate the absence of a label.

Many bin measurements are taken to determine the concentration of asample, and the absence or presence of a label is ascertained for eachbin measurement. Typically, 60,000 measurements or more can made in oneminute (e.g., in embodiments in which the bin size is 1 ms—for smallerbin sizes the number of measurements is correspondingly larger, e.g.,6,000,000 measurements per minute for a bin size of 10 microseconds).Thus, no single measurement is crucial and the method provides for ahigh margin of error. The bins that are determined not to contain alabel (“no” bins) are discounted and only the measurements made in thebins that are determined to contain label (“yes” bins) are accounted indetermining the concentration of the label in the processing sampleDiscounting measurements made in the “no” bins or bins that are devoidof label increases the signal to noise ratio and the accuracy of themeasurements. Thus, in some embodiments, determining the concentrationof a label in a sample comprises detecting the bin measurements thatreflect the presence of a label.

The signal to noise ratio or the sensitivity of the analyzer system canbe increased by minimizing the time that background noise is detectedduring a bin measurement in which a particle-label complex is detected.For example, in a bin measurement lasting 1 millisecond during which oneparticle-label complex is detected when passing across an interrogationspace within 250 microseconds, 750 microseconds of the 1 millisecond arespent detecting background noise emission. The signal to noise ratio canbe improved by decreasing the bin time. In some embodiments, the bintime is 1 millisecond. In other embodiments, the bin time is 750, 500,250 microseconds, 100 microseconds, 50 microseconds, 25 microseconds or10 microseconds. Other bin times are as described herein.

Other factors that affect measurements are the brightness or dimness ofthe fluorescent moiety, the flow rate, and the power of the laser.Various combinations of the relevant factors that allow for detection oflabel will be apparent to those of skill in the art. In someembodiments, the bin time is adjusted without changing the flow rate. Itwill be appreciated by those of skill in the art that as bin timedecreases, laser power output directed at the interrogation space mustincrease to maintain a constant total energy applied to theinterrogation space during the bin time. For example, if bin time isdecreased from 1000 microseconds to 250 microseconds, as a firstapproximation, laser power output must be increased approximatelyfour-fold. These settings allow for the detection of the same number ofphotons in a 250 μs as the number of photons counted during the 1000 μsgiven the previous settings, and allow for faster analysis of samplewith lower backgrounds and thus greater sensitivity. In addition, flowrates may be adjusted in order to speed processing of sample. Thesenumbers are merely exemplary, and the skilled practitioner can adjustthe parameters as necessary to achieve the desired result.

In some embodiments, the interrogation space encompasses the entirecross-section of the sample stream. When the interrogation spaceencompasses the entire cross-section of the sample stream, only thenumber of labels counted and the volume passing through a cross-sectionof the sample stream in a set length of time are needed to calculate theconcentration of the label in the processing sample. In someembodiments, the interrogation space can be defined to be smaller thanthe cross-sectional area of sample stream by, for example, theinterrogation space is defined by the size of the spot illuminated bythe laser beam. In some embodiments, the interrogation space can bedefined by adjusting the apertures 306 (FIG. 1A) or 358 and 359 (FIG.1B) of the analyzer and reducing the illuminated volume that is imagedby the objective lens to the detector. In the embodiments when theinterrogation space is defined to be smaller than the cross-sectionalarea of sample stream, the concentration of the label can be determinedby interpolation of the signal emitted by the complex from a standardcurve that is generated using one or more samples of known standardconcentrations. In yet other embodiments, the concentration of the labelcan be determined by comparing the measured particles to an internallabel standard. In embodiments when a diluted sample is analyzed, thedilution factor is accounted in calculating the concentration of themolecule of interest in the starting sample

As discussed above, when the interrogation space encompasses the entirecross-section of the sample stream, only the number of labels countedpassing through a cross-section of the sample stream in a set length oftime (bin) and the volume of sample that was interrogated in the bin areneeded to calculate the concentration the sample. The total number oflabels contained in the “yes” bins is determined and related to thesample volume represented by the total number of bins used in theanalysis to determine the concentration of labels in the processingsample Thus, in one embodiment, determining the concentration of a labelin a processing sample comprises determining the total number of labelsdetected “yes” bins and relating the total number of detected labels tothe total sample volume that was analyzed. The total sample volume thatis analyzed is the sample volume that is passed through the capillaryflow cell and across the interrogation space in a specified timeinterval. Alternatively, the concentration of the label complex in asample is determined by interpolation of the signal emitted by the labelin a number of bins from a standard curve that is generated bydetermining the signal emitted by labels in the same number of bins bystandard samples containing known concentrations of the label.

In some embodiments, the number of individual labels that are detectedin a bin is related to the relative concentration of the particle in theprocessing sample. At relatively low concentrations, for example atconcentrations below about 10-16 M the number of labels is proportionalto the photon signal that is detected in a bin. Thus, at lowconcentrations of label the photon signal is provided as a digitalsignal. At relatively higher concentrations, for example atconcentrations greater than about 10-16 M, the proportionality of photonsignal to a label is lost as the likelihood of two or more labelscrossing the interrogation space at about the same time and beingcounted as one becomes significant. Thus, in some embodiments,individual particles in a sample of a concentration greater than about10⁻¹⁶ M are resolved by decreasing the length of time of the binmeasurement.

Alternatively, in other embodiments, the total the photon signal that isemitted by a plurality of particles that are present in any one bin isdetected. These embodiments allow for single molecule detectors of theinvention wherein the dynamic range is at least 3, 3.5, 4, 4.5, 5.5, 6,6.5, 7, 7.5, 8, or more than 8 logs.

“Dynamic range,” as that term is used herein, refers to the range ofsample concentrations that may be quantitated by the instrument withoutneed for dilution or other treatment to alter the concentration ofsuccessive samples of differing concentrations, where concentrations aredetermined with an accuracy appropriate for the intended use. Forexample, if a microtiter plate contains a sample of 1 femtomolarconcentration for an analyte of interest in one well, a sample of 10,000femtomolar concentration for an analyte of interest in another well, anda sample of 100 femtomolar concentration for the analyte in a thirdwell, an instrument with a dynamic range of at least 4 logs and a lowerlimit of quantitation of 1 femtomolar is able to accurately quantitatethe concentration of all the samples without the need for furthertreatment to adjust concentration, e.g., dilution. Accuracy may bedetermined by standard methods, e.g., using a series of standards ofconcentrations that span the dynamic range and constructing a standardcurve Standard measures of fit of the resulting standard curve may beused as a measure of accuracy, e.g., an r² greater than about 0.7, 0.75,0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.

Increased dynamic range is achieved by altering the manner in which datafrom the detector is analyzed, and/or by the use of an attenuatorbetween the detector and the interrogation space. At the low end of therange, where processing sample is sufficiently dilute that eachdetection event, i.e., each burst of photons above a threshold level ina bin (the “event photons”), likely represents only one label, the datais analyzed to count detection events as single molecules. I.e., eachbin is analyzed as a simple “yes” or “no” for the presence of label, asdescribed above. For a more concentrated processing sample, where thelikelihood of two or more labels occupying a single bin becomessignificant, the number of event photons in a significant number of binsis found to be substantially greater than the number expected for asingle label, e.g., the number of event photons in a significant numberof bins corresponds to two-fold, three-fold, or more, than the number ofevent photons expected for a single label. For these samples, theinstrument changes its method of data analysis to one of integrating thetotal number of event photons for the bins of the processing sample.This total will be proportional to the total number of labels that werein all the bins. For an even more concentrated processing sample, wheremany labels are present in most bins, background noise becomes aninsignificant portion of the total signal from each bin, and theinstrument changes its method of data analysis to one of counting totalphotons per bin (including background). An even further increase indynamic range can be achieved by the use of an attenuator between theflow cell and the detector, when concentrations are such that theintensity of light reaching the detector would otherwise exceed thecapacity of the detector for accurately counting photons, i.e., saturatethe detector.

The instrument may include a data analysis system that receives inputfrom the detector and determines the appropriate analysis method for thesample being run, and outputs values based on such analysis. The dataanalysis system may further output instructions to use or not use anattenuator, if an attenuator is included in the instrument.

By utilizing such methods, the dynamic range of the instrument can bedramatically increased. Thus, in some embodiments, the instrument iscapable of measuring concentrations of samples over a dynamic range ofmore than about 1000 (3 log), 10,000 (4 log), 100,000 (5 log), 350,000(5.5 log), 1,000,000 (6 log), 3,500,000 (6.5 log), 10,000,000 (7 log),35,000,000 (7.5 log), or 100,000,000 (8 log). In some embodiments, theinstrument is capable of measuring concentrations of samples over adynamic range of more than about 100,000 (5 log). In some embodiments,the instrument is capable of measuring concentrations of samples over adynamic range of more than about 1,000,000 (6 log). In some embodiments,the instrument is capable of measuring concentrations of samples over adynamic range of more than about 10,000,000 (7 log). In someembodiments, the instrument is capable of measuring the concentrationsof samples over a dynamic range of from about 1-10 femtomolar to atleast about 1000; 10,000; 100,000; 350,000; 1,000,000; 3,500,000;10,000,000, or 35,000,000 femtomolar. In some embodiments, theinstrument is capable of measuring the concentrations of samples over adynamic range of from about 1-10 femtomolar to at least about 10,000femtomolar. In some embodiments, the instrument is capable of measuringthe concentrations of samples over a dynamic range of from about 1-10femtomolar to at least about 100,000 femtomolar. In some embodiments,the instrument is capable of measuring the concentrations of samplesover a dynamic range of from about 1-10 femtomolar to at least about1,000,000 femtomolar. In some embodiments, the instrument is capable ofmeasuring the concentrations of samples over a dynamic range of fromabout 1-10 femtomolar to at least about 10,000,000.

In some embodiments, an analyzer or analyzer system of the invention iscapable of detecting an analyte, e.g., a biomarker at a limit ofdetection of less than 1 nanomolar, or 1 picomolar, or 1 femtomolar, or1 attomolar, or 1 zeptomolar. In some embodiments, the analyzer oranalyzer system is capable of detecting a change in concentration of theanalyte, or of multiple analytes, e.g., a biomarker or biomarkers, fromone sample to another sample of less than about 0.1, 1, 2, 5, 10, 20,30, 40, 50, 60, or 80% when the biomarker is present at a concentrationof less than 1 nanomolar, or 1 picomolar, or 1 femtomolar, or 1attomolar, or 1 zeptomolar, in the samples, and when the size of each ofthe sample is less than about 100, 50, 40, 30, 20, 10, 5, 2, 1, 0.1,0.01, 0.001, or 0.0001 ul. In some embodiments, the analyzer or analyzersystem is capable of detecting a change in concentration of the analytefrom a first sample to a second sample of less than about 20%, when theanalyte is present at a concentration of less than about 1 picomolar,and when the size of each of the samples is less than about 50 μl. Insome embodiments, the analyzer or analyzer system is capable ofdetecting a change in concentration of the analyte from a first sampleto a second sample of less than about 20%, when the analyte is presentat a concentration of less than about 100 femtomolar, and when the sizeof each of the samples is less than about 50 μl. In some embodiments,the analyzer or analyzer system is capable of detecting a change inconcentration of the analyte from a first sample to a second sample ofless than about 20%, when the analyte is present at a concentration ofless than about 50 femtomolar, and when the size of each of the samplesis less than about. 50 μl. In some embodiments, the analyzer or analyzersystem is capable of detecting a change in concentration of the analytefrom a first sample to a second sample of less than about 20%, when theanalyte is present at a concentration of less than about 5 femtomolar,and when the size of each of the samples is less than about 50 μl. Insome embodiments, the analyzer or analyzer system is capable ofdetecting a change in concentration of the analyte from a first sampleto a second sample of less than about 20%, when the analyte is presentat a concentration of less than about 5 femtomolar, and when the size ofeach of the samples is less than about 5 μl. In some embodiments, theanalyzer or analyzer system is capable of detecting a change inconcentration of the analyte from a first sample to a second sample ofless than about 20%, when the analyte is present at a concentration ofless than about 1 femtomolar, and when the size of each of the samplesis less than about 5 μl.

V. Instruments and Systems Suitable for Highly Sensitive Analysis ofTroponin

The methods of the invention utilize analytical instruments of highsensitivity, e.g., single molecule detectors. Such single moleculedetectors include embodiments as hereinafter described.

A. Apparatus/System

In one aspect, the methods described herein utilize an analyzer systemcapable of detecting a single particle in a sample. In one embodiment,the analyzer system is capable of single particle detection of afluorescently labeled particle wherein the analyzer system detectsenergy emitted by an excited fluorescent label in response to exposureby an electromagnetic radiation source when the single particle ispresent in an interrogation space defined within a capillary flow cellfluidly connected to the sampling system of the analyzer system. In afurther embodiment of the analyzer system, the single particle movesthrough the interrogation space of the capillary flow cell by means of amotive force. In another embodiment of the analyzer system, an automaticsampling system may be included in the analyzer system for introducingthe sample into the analyzer system. In another embodiment of theanalyzer system, a sample preparation system may be included in theanalyzer system for preparing a sample. In a further embodiment, theanalyzer system may contain a sample recovery system for recovering atleast a portion of the sample after analysis is complete.

In one aspect, the analyzer system consists of an electromagneticradiation source for exciting a single particle labeled with afluorescent label. In one embodiment, the electromagnetic radiationsource of the analyzer system is a laser. In a further embodiment, theelectromagnetic radiation source is a continuous wave laser.

In a typical embodiment, the electromagnetic radiation source excites afluorescent moiety attached to a label as the label passes through theinterrogation space of the capillary flow cell. In some embodiments, thefluorescent label moiety includes one or more fluorescent dye molecules.In some embodiments, the fluorescent label moiety is a quantum dot. Anyfluorescent moiety as described herein may be used in the label.

A label is exposed to electromagnetic radiation when the label passesthrough an interrogation space located within the capillary flow cell.The interrogation space is typically fluidly connected to a samplingsystem. In some embodiments the label passes through the interrogationspace of the capillary flow cell due to a motive force to advance thelabel through the analyzer system. The interrogation space is positionedsuch that it receives electromagnetic radiation emitted from theradiation source. In some embodiments, the sampling system is anautomated sampling system capable of sampling a plurality of sampleswithout intervention from a human operator.

The label passes through the interrogation space and emits a detectableamount of energy when excited by the electromagnetic radiation source.In one embodiment, an electromagnetic radiation detector is operablyconnected to the interrogation space. The electromagnetic radiationdetector is capable of detecting the energy emitted by the label, e.g.,by the fluorescent moiety of the label.

In a further embodiment of the analyzer system, the system furtherincludes a sample preparation mechanism where a sample may be partiallyor completely prepared for analysis by the analyzer system. In someembodiments of the analyzer system, the sample is discarded after it isanalyzed by the system. In other embodiments, the analyzer systemfurther includes a sample recovery mechanism whereby at least a portion,or alternatively all or substantially all, of the sample may berecovered after analysis. In such an embodiment, the sample can bereturned to the origin of the sample. In some embodiments, the samplecan be returned to microtiter wells on a sample microtiter plate. Theanalyzer system typically further consists of a data acquisition systemfor collecting and reporting the detected signal

B. Single Particle Analyzer

As shown in FIG. 1A, described herein is one embodiment of an analyzersystem 300. The analyzer system 300 includes an electromagneticradiation source 301, a mirror 302, a lens 303, a capillary flow cell313, a microscopic objective lens 305, an aperture 306, a detector lens307, a detector filter 308, a single photon detector 309, and aprocessor 310 operatively connected to the detector.

In operation the electromagnetic radiation source 301 is aligned so thatits output 311 is reflected off of a front surface 312 of mirror 302.The lens 303 focuses the beam 311 onto a single interrogation space (anillustrative example of an interrogation space 314 is shown in FIG. 2A)in the capillary flow cell 313. The microscope objective lens 305collects light from sample particles and forms images of the beam ontothe aperture 306. The aperture 306 affects the fraction of light emittedby the specimen in the interrogation space of the capillary flow cell313 that can be collected. The detector lens 307 collects the lightpassing through the aperture 306 and focuses the light onto an activearea of the detector 309 after it passes through the detector filters308. The detector filters 308 minimize aberrant noise signals due tolight scatter or ambient light while maximizing the signal emitted bythe excited fluorescent moiety bound to the particle. The processor 310processes the light signal from the particle according to the methodsdescribed herein.

In one embodiment, the microscope objective lens 305 is a high numericalaperture microscope objective. As used herein, “high numerical aperturelens” include a lens with a numerical aperture of equal to or greaterthan 0.6. The numerical aperture is a measure of the number of highlydiffracted image-forming light rays captured by the objective. A highernumerical aperture allows increasingly oblique rays to enter theobjective lens and thereby produce a more highly resolved image.Additionally, the brightness of an image increases with a highernumerical aperture. High numerical aperture lenses are commerciallyavailable from a variety of vendors, and any one lens having a numericalaperture of equal to or greater than approximately 0.6 may be used inthe analyzer system. In some embodiments, the lens has a numericalaperture of about 0.6 to about 1.3. In some embodiments, the lens has anumerical aperture of about 0.6 to about 1.0. In some embodiments, thelens has a numerical aperture of about 0.7 to about 1.2. In someembodiments, the lens has a numerical aperture of about 0.7 to about1.0. In some embodiments, the lens has a numerical aperture of about 0.7to about 0.9. In some embodiments, the lens has a numerical aperture ofabout 0.8 to about 1.3. In some embodiments, the lens has a numericalaperture of about 0.8 to about 1.2. In some embodiments, the lens has anumerical aperture of about 0.8 to about 1.0. In some embodiments, thelens has a numerical aperture of at least about 0.6. In someembodiments, the lens has a numerical aperture of at least about 0.7. Insome embodiments, the lens has a numerical aperture of at least about0.8. In some embodiments, the lens has a numerical aperture of at leastabout 0.9. In some embodiments, the lens has a numerical aperture of atleast about 1.0. In some embodiments, the aperture of the microscopeobjective lens 305 is approximately 1.25. In an embodiment where amicroscope objective lens 305 of 0.8 is used, a Nikon 60X/0.8 NAAchromat lens (Nikon, Inc., USA) can be used.

In some embodiments, the electromagnetic radiation source 301 is a laserthat emits light in the visible spectrum. In all embodiments, theelectromagnetic radiation source is set such that wavelength of thelaser is set such that it is of a sufficient wavelength to excite thefluorescent label attached to the particle. In some embodiments, thelaser is a continuous wave laser with a wavelength of 639 nm. In otherembodiments, the laser is a continuous wave laser with a wavelength of532 nm. In other embodiments, the laser is a continuous wave laser witha wavelength of 422 nm. In other embodiments, the laser is a continuouswave laser with a wavelength of 405 nm. Any continuous wave laser with awavelength suitable for exciting a fluorescent moiety as used in themethods and compositions of the invention may be used without departingfrom the scope of the invention.

In a single particle analyzer system 300, as each particle passesthrough the beam 311 of the electromagnetic radiation source, theparticle enters into an excited state. When the particle relaxes fromits excited state, a detectable burst of light is emitted. Theexcitation-emission cycle is repeated many times by each particle in thelength of time it takes for it to pass through the beam allowing theanalyzer system 300 to detect tens to thousands of photons for eachparticle as it passes through an interrogation space 314. Photonsemitted by fluorescent particles are registered by the detector 309(FIG. 1A) with a time delay indicative of the time for the particlelabel complex to pass through the interrogation space. The photonintensity is recorded by the detector 309 and sampling time is dividedinto bins, which are uniform, arbitrary, time segments with freelyselectable time channel widths. The number of signals contained in eachbin evaluated. One or a combination of several statistical analyticalmethods are employed in order to determine when a particle is present.Such methods include determining the baseline noise of the analyzersystem and setting a signal strength for the fluorescent label at astatistical level above baseline noise to eliminate false positivesignals from the detector.

The electromagnetic radiation source 301 is focused onto a capillaryflow cell 313 of the analyzer system 300 where the capillary flow cell313 is fluidly connected to the sample system. An interrogation space314 is shown in FIG. 2A. The beam 311 from the continuous waveelectromagnetic radiation source 301 of FIG. 1A is optically focused toa specified depth within the capillary flow cell 313. The beam 311 isdirected toward the sample-filled capillary flow cell 313 at an angleperpendicular to the capillary flow cell 313. The beam 311 is operatedat a predetermined wavelength that is selected to excite a particularfluorescent label used to label the particle of interest. The size orvolume of the interrogation space 314 is determined by the diameter ofthe beam 311 together with the depth at which the beam 311 is focused.Alternatively, the interrogation space can be determined by running acalibration sample of known concentration through the analyzer system.

When single molecules are detected in the sample concentration, the beamsize and the depth of focus required for single molecule detection areset and thereby define the size of the interrogation space 314. Theinterrogation space 314 is set such that, with an appropriate sampleconcentration, only one particle is present in the interrogation space314 during each time interval over which time observations are made. Itwill be appreciated that the detection interrogation volume as definedby the beam is not perfectly spherically shaped, and typically is a“bow-tie” shape. However, for the purposes of definition, “volumes” ofinterrogation spaces are defined herein as the volume encompassed by asphere of a diameter equal to the focused spot diameter of the beam. Thefocused spot of the beam 311 may have various diameters withoutdeparting from the scope of the present invention. In some embodiments,the diameter of the focused spot of the beam is about 1 to about 5, 10,15, or 20 microns, or about 5 to about 10, 15, or 20 microns, or about10 to about 20 microns, or about 10 to about 15 microns. In someembodiments, the diameter of the focused spot of the beam is about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20microns. In some embodiments, the diameter of the focused spot of thebeam is about 5 microns. In some embodiments, the diameter of thefocused spot of the beam is about 10 microns. In some embodiments, thediameter of the focused spot of the beam is about 12 microns. In someembodiments, the diameter of the focused spot of the beam is about 13microns. In some embodiments, the diameter of the focused spot of thebeam is about 14 microns. In some embodiments, the diameter of thefocused spot of the beam is about 15 microns. In some embodiments, thediameter of the focused spot of the beam is about 16 microns. In someembodiments, the diameter of the focused spot of the beam is about 17microns. In some embodiments, the diameter of the focused spot of thebeam is about 18 microns. In some embodiments, the diameter of thefocused spot of the beam is about 19 microns. In some embodiments, thediameter of the focused spot of the beam is about 20 microns.

In an alternate embodiment of the single particle analyzer system, morethan one electromagnetic radiation source can be used to exciteparticles labeled with fluorescent labels of different wavelengths. Inanother alternate embodiment, more than one interrogation space in thecapillary flow cell can be used. In another alternate embodiment,multiple detectors can be employed to detect different emissionwavelengths from the fluorescent labels. An illustration incorporatingeach of these alternative embodiments of an analyzer system is shown inFIG. 1B. These embodiments are incorporated by reference from previousU.S. patent application Ser. No. 11/048,660.

In some embodiments of the analyzer system 300, a motive force isrequired to move a particle through the capillary flow cell 313 of theanalyzer system 300. In one embodiment, the motive force can be a formof pressure. The pressure used to move a particle through the capillaryflow cell can be generated by a pump. In some embodiments, a Scivex,Inc. HPLC pump can be used. In some embodiments where a pump is used asa motive force, the sample can pass through the capillary flow cell at arate of 1 μL/min to about 20 μL/min, or about 5 μL/min to about 20μL/min. In some embodiments, the sample can pass through the capillaryflow cell at a rate of about 5 μL/min. In some embodiments, the samplecan pass through the capillary flow cell at a rate of about 10 μL/min.In some embodiments, the sample can pass through the capillary flow cellat a rate of about 15 μL/min. In some embodiments, the sample can passthrough the capillary flow cell at a rate of about 20 μL/min. In someembodiments, an electrokinetic force can be used to move the particlethrough the analyzer system. Such a method has been previously disclosedand is incorporated by reference from previous U.S. patent applicationSer. No. 11/048,660.

In one aspect of the analyzer system 300, the detector 309 of theanalyzer system detects the photons emitted by the fluorescent label. Inone embodiment, the photon detector is a photodiode. In a furtherembodiment, the detector is an avalanche photodiode detector. In someembodiments, the photodiodes can be silicon photodiodes with awavelength detection of 190 nm and 1100 nm. When germanium photodiodesare used, the wavelength of light detected is between 400 nm to 1700 nm.In other embodiments, when an indium gallium arsenide photodiode isused, the wavelength of light detected by the photodiode is between 800nm and 2600 nm. When lead sulfide photodiodes are used as detectors, thewavelength of light detected is between 1000 nm and 3500 nm.

In some embodiments, the optics of the electromagnetic radiation source301 and the optics of the detector 309 are arranged in a conventionaloptical arrangement. In such an arrangement, the electromagneticradiation source and the detector are aligned on different focal planes.The arrangement of the laser and the detector optics of the analyzersystem as shown in FIGS. 1A and 1B is that of a conventional opticalarrangement.

In some embodiments, the optics of the electromagnetic radiation sourceand the optics of the detector are arranged in a confocal opticalarrangement. In such an arrangement, the electromagnetic radiationsource 301 and the detector 309 are aligned on the same focal plane. Theconfocal arrangement renders the analyzer more robust because theelectromagnetic radiation source 301 and the detector optics 309 do notneed to be realigned if the analyzer system is moved. This arrangementalso makes the use of the analyzer more simplified because it eliminatesthe need to realign the components of the analyzer system. The confocalarrangement for the analyzer 300 (FIG. 1A) and the analyzer 355 (FIG.1B) are shown in FIGS. 3A and 3B respectively. FIG. 3A shows that thebeam 311 from an electromagnetic radiation source 301 is focused by themicroscope objective 315 to form one interrogation space 314 (FIG. 2A)within the capillary flow cell 313. A dichroic mirror 316, whichreflects laser light but passes fluorescent light, is used to separatethe fluorescent light from the laser light. Filter 317 that ispositioned in front of the detector eliminates any non-fluorescent lightat the detector. In some embodiments, an analyzer system configured in aconfocal arrangement can comprise two or more interrogations spaces.Such a method has been previously disclosed and is incorporated byreference from previous U.S. patent application Ser. No. 11/048,660.

The laser can be a tunable dye laser, such as a helium-neon laser. Thelaser can be set to emit a wavelength of 632.8 nm. Alternatively, thewavelength of the laser can be set to emit a wavelength of 543.5 nm or1523 nm. Alternatively, the electromagnetic laser can be an argon ionlaser. In such an embodiment, the argon ion laser can be operated as acontinuous gas laser at about 25 different wavelengths in the visiblespectrum, the wavelength set between 408.9 and 686.1 nm but at itsoptimum performance set between 488 and 514.5 nm.

1 Electromagnetic Radiation Source

In some embodiments of the analyzer system a chemiluminescent label maybe used. In such an embodiment, it may not be necessary to utilize an EMsource for detection of the particle. In another embodiment, theextrinsic label or intrinsic characteristic of the particle is alight-interacting label or characteristic, such as a fluorescent labelor a light-scattering label. In such an embodiment, a source of EMradiation is used to illuminate the label and/or the particle. EMradiation sources for excitation of fluorescent labels are preferred.

In some embodiments, the analyzer system consists of an electromagneticradiation source 301. Any number of radiation sources may be used in anyone analyzer system 300 without departing from the scope of theinvention. Multiple sources of electromagnetic radiation have beenpreviously disclosed and are incorporated by reference from previousU.S. patent application Ser. No. 11/048,660. In some embodiments, allthe continuous wave electromagnetic (EM) radiation sources emitelectromagnetic radiation at the same wavelengths. In other embodiments,different sources emit different wavelengths of EM radiation.

In one embodiment, the EM source(s) 301, 351, 352 are continuous wavelasers producing wavelengths of between 200 nm and 1000 nm. Such EMsources have the advantage of being small, durable and relativelyinexpensive. In addition, they generally have the capacity to generatelarger fluorescent signals than other light sources. Specific examplesof suitable continuous wave EM sources include, but are not limited to:lasers of the argon, krypton, helium-neon, helium-cadmium types, as wellas, tunable diode lasers (red to infrared regions), each with thepossibility of frequency doubling. The lasers provide continuousillumination with no accessory electronic or mechanical devices, such asshutters, to interrupt their illumination. In an embodiment where acontinuous wave laser is used, an electromagnetic radiation source of 3mW may be of sufficient energy to excite a fluorescent label. A beamfrom a continuous wave laser of such energy output may be between 2 to 5μm in diameter. The time of exposure of the particle to laser beam inorder to be exposed to 3 mW may be a time period of about 1 msec. Inalternate embodiments, the time of exposure to the laser beam may beequal to or less than about 500 μsec. In an alternate embodiment, thetime of exposure may be equal to or less than about 100 μsec. In analternate embodiment, the time of exposure may be equal to or less thanabout 50 μsec. In an alternate embodiment, the time of exposure may beequal to or less than about 10 μsec.

LEDs are another low-cost, high reliability illumination source. Recentadvances in ultra-bright LEDs and dyes with high absorptioncross-section and quantum yield support the applicability of LEDs tosingle particle detection. Such lasers could be used alone or incombination with other light sources such as mercury arc lamps,elemental arc lamps, halogen lamps, arc discharges, plasma discharges,light-emitting diodes, or combination of these.

In other embodiments, the EM source could be in the form of a pulse wavelaser. In such an embodiment, the pulse size of the laser is animportant factor. In such an embodiment, the size, focus spot, and thetotal energy emitted by the laser is important and must be of sufficientenergy as to be able to excite the fluorescent label. When a pulse laseris used, a pulse of longer duration may be required. In some embodimentsa laser pulse of 2 nanoseconds may be used. In some embodiments a laserpulse of 5 nanoseconds may be used. In some embodiments a pulse ofbetween 2 to 5 nanoseconds may be used.

The optimal laser intensity depends on the photo bleachingcharacteristics of the single dyes and the length of time required totraverse the interrogation space (including the speed of the particle,the distance between interrogation spaces if more than one is used andthe size of the interrogation space(s)). To obtain a maximal signal, itis desirable to illuminate the sample at the highest intensity whichwill not result in photo bleaching a high percentage of the dyes. Thepreferred intensity is one such that no more that 5% of the dyes arebleached by the time the particle has traversed the interrogation space.

The power of the laser is set depending on the type of dye moleculesthat need to be stimulated and the length of time the dye molecules arestimulated, and/or the speed with which the dye molecules pass throughthe capillary flow cell. Laser power is defined as the rate at whichenergy is delivered by the beam and is measured in units ofJoules/second, or Watts. It will be appreciated that the greater thepower output of the laser, the shorter the time that the laserilluminates the particle may be, while providing a constant amount ofenergy to the interrogation space while the particle is passing throughthe space. Thus, in some embodiments, the combination of laser power andtime of illumination is such that the total energy received by theinterrogation space during the time of illumination is more than about0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, or 100 microJoule. In some embodiments, thecombination of laser power and time of illumination is such that thetotal energy received by the interrogation space during the time ofillumination is less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or 110 microJoule.In some embodiments, the combination of laser power and time ofillumination is such that the total energy received by the interrogationspace during the time of illumination is between about 0.1 and 100microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 1and 100 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 1and 50 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 2and 50 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 3and 60 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 3and 50 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 3and 40 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 3and 30 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is about 1microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 3microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 5microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 10microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 15microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 20microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 30microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 40microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 50microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 60microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 70microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 80microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 90microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 100microJoule.

In some embodiments, the laser power output is set to at least about 1mW, 2 mW, 3 mW, 4 mW, 5 mW, 6, mw, 7 mW, 8 mW, 9 mW, 10 mW, 13 mW, 15mW, 20 mW, 25 mW, 30 mW, 40 mW, 50 mW, 60 mW, 70 mW, 80 mW, 90 mW, 100mW, or more than 100 mW. In some embodiments, the laser power output isset to at least about 1 mW. In some embodiments, the laser power outputis set to at least about 3 mW. In some embodiments, the laser poweroutput is set to at least about 5 mW. In some embodiments, the laserpower output is set to at least about 10 mW. In some embodiments, thelaser power output is set to at least about 20 mW. In some embodiments,the laser power output is set to at least about 30 mW. In someembodiments, the laser power output is set to at least about 40 mW. Insome embodiments, the laser power output is set to at least about 50 mW.In some embodiments, the laser power output is set to at least about 60mW. In some embodiments, the laser power output is set to at least about90 mW.

The time that the laser illuminates the interrogation space can be setto no less than about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 150, 200, 150, 300, 350, 400, 450, 500, 600, 700, 800, 900, or1000 microseconds. The time that the laser illuminates the interrogationspace can be set to no more than about 2, 3, 4, 5, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 150, 300, 350, 400, 450, 500, 600,700, 800, 900, 1000, 1500, or 2000 microseconds. The time that the laserilluminates the interrogation space can be set between about 1 and 1000microseconds. The time that the laser illuminates the interrogationspace can be set between about 5 and 500 microseconds. The time that thelaser illuminates the interrogation space can be set between about 5 and100 microseconds. The time that the laser illuminates the interrogationspace can be set between about 10 and 100 microseconds. The time thatthe laser illuminates the interrogation space can be set between about10 and 50 microseconds. The time that the laser illuminates theinterrogation space can be set between about 10 and 20 microseconds. Thetime that the laser illuminates the interrogation space can be setbetween about 5 and 50 microseconds. The time that the laser illuminatesthe interrogation space can be set between about 1 and 100 microseconds.In some embodiments, the time that the laser illuminates theinterrogation space is about 1 microsecond. In some embodiments, thetime that the laser illuminates the interrogation space is about 5microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 10 microseconds. In some embodiments,the time that the laser illuminates the interrogation space is about 25microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 50 microseconds. In some embodiments,the time that the laser illuminates the interrogation space is about 100microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 250 microseconds. In some embodiments,the time that the laser illuminates the interrogation space is about 500microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 1000 microseconds.

For example, the time that the laser illuminates the interrogation spacecan be set to 1 millisecond, 250 microseconds, 100 microseconds, 50microseconds, 25 microseconds or 10 microseconds with a laser thatprovides a power output of 3 mW, 4 mw, 5 mW, or more than 5 mW. In someembodiments, a label is illuminated with a laser that provides a poweroutput of 3 mW and illuminates the label for about 1000 microseconds. Inother embodiments, a label is illuminated for less than 1000milliseconds with a laser providing a power output of not more thanabout 20 mW. In other embodiments, the label is illuminated with a laserpower output of 20 mW for less than or equal to about 250 microseconds.In some embodiments, the label is illuminated with a laser power outputof about 5 mW for less than or equal to about 1000 microseconds.

2. Capillary Flow Cell

The capillary flow cell is fluidly connected to the sample system. Inone embodiment, the interrogation space 314 of an analyzer system, isdetermined by the cross sectional area of the corresponding beam 311 andby a segment of the beam within the field of view of the detector 309.In one embodiment of the analyzer system, the interrogation space 314has a volume, as defined herein, of between about between about 0.01 and500 pL, or between about 0.01 pL and 100 pL, or between about 0.01 pLand 10 pL, or between about 0.01 pL and 1 pL, or between about 0.01 pLand 0.5 pL, or between about 0.02 pL and about 300 pL, or between about0.02 pL and about 50 pL or between about 0.02 pL and about 5 pL orbetween about 0.02 pL and about 0.5 pL or between about 0.02 pL andabout 2 pL, or between about 0.05 pL and about 50 pL, or between about0.05 pL and about 5 pL, or between about 0.05 pL and about 0.5 pL, orbetween about 0.05 pL and about 0.2 pL, or between about 0.1 pL andabout 25 pL. In some embodiments, the interrogation space has a volumebetween about 0.01 pL and 10 pL. In some embodiments, the interrogationspace 314 has a volume between about 0.01 pL and 1 pL. In someembodiments, the interrogation space 314 has a volume between about 0.02pL and about 5 pL. In some embodiments, the interrogation space 314 hasa volume between about 0.02 pL and about 0.5 pL. In some embodiments,the interrogation space 314 has a volume between about 0.05 pL and about0.2 pL. In some embodiments, the interrogation space 314 has a volume ofabout 0.1 pL. Other useful interrogation space volumes are as describedherein. It should be understood by one skilled in the art that theinterrogation space 314 can be selected for maximum performance of theanalyzer. Although very small interrogation spaces have been shown tominimize the background noise, large interrogation spaces have theadvantage that low concentration samples can be analyzed in a reasonableamount of time. In embodiments in which two interrogation spaces 370 and371 are used, volumes such as those described herein for a singleinterrogation space 314 may be used.

In one embodiment of the present invention, the interrogation spaces arelarge enough to allow for detection of particles at concentrationsranging from about 1000 femtomolar (fM) to about 1 zeptomolar (zM). Inone embodiment of the present invention, the interrogation spaces arelarge enough to allow for detection of particles at concentrationsranging from about 1000 fM to about 1 attomolar (aM). In one embodimentof the present invention, the interrogation spaces are large enough toallow for detection of particles at concentrations ranging from about 10fM to about 1 attomolar (aM). In many cases, the large interrogationspaces allow for the detection of particles at concentrations of lessthan about 1 fM without additional pre-concentration devices ortechniques. One skilled in the art will recognize that the mostappropriate interrogation space size depends on the brightness of theparticles to be detected, the level of background signal, and theconcentration of the sample to be analyzed.

The size of the interrogation space 314 can be limited by adjusting theoptics of the analyzer. In one embodiment, the diameter of the beam 311can be adjusted to vary the volume of the interrogation space 314. Inanother embodiment, the field of view of the detector 309 can be varied.Thus, the source 301 and the detector 309 can be adjusted so that singleparticles will be illuminated and detected within the interrogationspace 314. In another embodiment, the width of aperture 306 (FIG. 1A)that determine the field of view of the detector 309 is variable. Thisconfiguration allows for altering the interrogation space, in near realtime, to compensate for more or less concentrated samples, ensuring alow probability of two or more particles simultaneously being within aninterrogation space Similar alterations for two or more interrogationspaces, 370 and 371, may performed.

In another embodiment, the interrogation space can be defined throughthe use of a calibration sample of known concentration that is passedthrough the capillary flow cell prior to the actual sample being tested.When only one single particle is detected at a time in the calibrationsample as the sample is passing through the capillary flow cell, thedepth of focus together with the diameter of the beam of theelectromagnetic radiation source determines the size of theinterrogation space in the capillary flow cell.

Physical constraints to the interrogation spaces can also be provided bya solid wall. In one embodiment, the wall is one or more of the walls ofa flow cell 313 (FIG. 2A), when the sample fluid is contained within acapillary. In one embodiment, the cell is made of glass, but othersubstances transparent to light in the range of about 200 to about 1,000nm or higher, such as quartz, fused silica, and organic materials suchas Teflon, nylon, plastics, such as polyvinylchloride, polystyrene, andpolyethylene, or any combination thereof, may be used without departingfrom the scope of the present invention. Although other cross-sectionalshapes (e.g., rectangular, cylindrical) may be used without departingfrom the scope of the present invention, in one embodiment the capillaryflow cell 313 has a square cross section. In another embodiment, theinterrogation space may be defined at least in part by a channel (notshown) etched into a chip (not shown) Similar considerations apply toembodiments in which two interrogation spaces are used (370 and 371 inFIG. 2B).

The interrogation space is bathed in a fluid. In one embodiment, thefluid is aqueous. In other embodiments, the fluid is non-aqueous or acombination of aqueous and non-aqueous fluids. In addition the fluid maycontain agents to adjust pH, ionic composition, or sieving agents, suchas soluble macroparticles or polymers or gels. It is contemplated thatvalves or other devices may be present between the interrogation spacesto temporarily disrupt the fluid connection. Interrogation spacestemporarily disrupted are considered to be connected by fluid.

In another embodiment of the invention, an interrogation space is thesingle interrogation space present within the flow cell 313 which isconstrained by the size of a laminar flow of the sample material withina diluent volume, also called sheath flow. In these and otherembodiments, the interrogation space can be defined by sheath flow aloneor in combination with the dimensions of the illumination source or thefield of view of the detector. Sheath flow can be configured in numerousways, including: The sample material is the interior material in aconcentric laminar flow, with the diluent volume in the exterior; thediluent volume is on one side of the sample volume; the diluent volumeis on two sides of the sample material; the diluent volume is onmultiple sides of the sample material, but not enclosing the samplematerial completely; the diluent volume completely surrounds the samplematerial; the diluent volume completely surrounds the sample materialconcentrically; the sample material is the interior material in adiscontinuous series of drops and the diluent volume completelysurrounds each drop of sample material.

In some embodiments, single molecule detectors of the invention compriseno more than one interrogation space. In some embodiments, multipleinterrogation spaces are used. Multiple interrogation spaces have beenpreviously disclosed and are incorporated by reference from U.S. patentapplication Ser. No. 11/048,660. One skilled in the art will recognizethat in some cases the analyzer will contain 2, 3, 4, 5, 6 or moredistinct interrogation spaces.

3. Motive Force

In one embodiment of the analyzer system, the particles are movedthrough the interrogation space by a motive force. In some embodiments,the motive force for moving particles is pressure. In some embodiments,the pressure is supplied by a pump, and air pressure source, a vacuumsource, a centrifuge, or a combination thereof. In some embodiments, themotive force for moving particles is an electrokinetic force. The use ofan electrokinetic force as a motive force has been previously disclosedin a prior application and is incorporated by reference from U.S. patentapplication Ser. No. 11/048,660.

In one embodiment, pressure can be used as a motive force to moveparticles through the interrogation space of the capillary flow cell. Ina further embodiment, pressure is supplied to move the sample by meansof a pump. Suitable pumps are known in the art. In one embodiment, pumpsmanufactured for HPLC applications, such as those made by Scivax, Inc.can be used as a motive force. In other embodiments, pumps manufacturedfor microfluidics applications can be used when smaller volumes ofsample are being pumped. Such pumps are described in U.S. Pat. Nos.5,094,594, 5,730,187, 6,033,628, and 6,533,553, which discloses deviceswhich can pump fluid volumes in the nanoliter or picoliter range.Preferably all materials within the pump that come into contact withsample are made of highly inert materials, e.g., polyetheretherketone(PEEK), fused silica, or sapphire.

A motive force is necessary to move the sample through the capillaryflow cell to push the sample through the interrogation space foranalysis. A motive force is also required to push a flushing samplethrough the capillary flow cell after the sample has been passedthrough. A motive force is also required to push the sample back outinto a sample recovery vessel, when sample recovery is employed Standardpumps come in a variety of sizes, and the proper size may be chosen tosuit the anticipated sample size and flow requirements. In someembodiments, separate pumps are used for sample analysis and forflushing of the system. The analysis pump may have a capacity ofapproximately 0.000001 mL to approximately 10 mL, or approximately 0.001mL to approximately 1 mL, or approximately 0.01 mL to approximately 0.2mL, or approximately 0.005, 0.01, 0.05, 0.1, or 0.5 mL. Flush pumps maybe of larger capacity than analysis pumps. Flush pumps may have a volumeof about 0.01 mL to about 20 mL, or about 0.1 mL to about 10 mL, orabout 0.1 mL to about 2 mL, or about or about 0.05, 0.1, 0.5, 1, 5, or10 mL. These pump sizes are illustrative only, and those of skill in theart will appreciate that the pump size may be chosen according to theapplication, sample size, viscosity of fluid to be pumped, tubingdimensions, rate of flow, temperature, and other factors well known inthe art. In some embodiments, pumps of the system are driven by steppermotors, which are easy to control very accurately with a microprocessor.

In preferred embodiments, the flush and analysis pumps are used inseries, with special check valves to control the direction of flow. Theplumbing is designed so that when the analysis pump draws up the maximumsample, the sample does not reach the pump itself. This is accomplishedby choosing the ID and length of the tubing between the analysis pumpand the analysis capillary such that the tubing volume is greater thanthe stroke volume of the analysis pump.

4. Detectors

In one embodiment, light (e.g., light in the ultra-violet, visible orinfrared range) emitted by a fluorescent label after exposure toelectromagnetic radiation is detected. The detector 309 (FIG. 1A), ordetectors (364, 365, FIG. 1B), is capable of capturing the amplitude andduration of photon bursts from a fluorescent label-moiety complex, andfurther converting the amplitude and duration of the photon burst toelectrical signals. Detection devices such as CCD cameras, video inputmodule cameras, and Streak cameras can be used to produce images withcontiguous signals. In another embodiment, devices such as a bolometer,a photodiode, a photodiode array, avalanche photodiodes, andphotomultipliers which produce sequential signals may be used. Anycombination of the aforementioned detectors may also be used. In oneembodiment, avalanche photodiodes are used for detecting photons.

Using specific optics between an interrogation space 314 (FIG. 2A) andits corresponding detector 309 (FIG. 1A), several distinctcharacteristics of the emitted electromagnetic radiation can be detectedincluding: emission wavelength, emission intensity, burst size, burstduration, and fluorescence polarization. In some embodiments, thedetector 309 is a photodiode that is used in reverse bias. A photodiodeset in reverse bias usually has an extremely high resistance. Thisresistance is reduced when light of an appropriate frequency shines onthe P/N junction. Hence, a reverse biased diode can be used as adetector by monitoring the current running through it. Circuits based onthis effect are more sensitive to light than ones based on zero bias.

In one embodiment of the analyzer system, the photodiode can be anavalanche photodiode, which can be operated with much higher reversebias than conventional photodiodes, thus allowing each photo-generatedcarrier to be multiplied by avalanche breakdown, resulting in internalgain within the photodiode, which increases the effective responsiveness(sensitivity) of the device. The choice of photodiode is determined bythe energy or emission wavelength emitted by the fluorescently labeledparticle. In some embodiments, the photodiode is a silicon photodiodethat detects energy in the range of 190-1100 nm; in another embodimentthe photodiode is a germanium photodiode that detects energy in therange of 800-1700 nm; in another embodiment the photodiode is an indiumgallium arsenide photodiode that detects energy in the range of 800-2600nm; and in yet other embodiments, the photodiode is a lead sulfidephotodiode that detects energy in the range of between less than 1000 nmto 3500 nm. In some embodiments, the avalanche photodiode is asingle-photon detector designed to detect energy in the 400 nm to 1100nm wavelength range. Single photon detectors are commercially available(for example Perkin Elmer, Wellesley, Mass.).

In some embodiments the detector is a avalanche photodiode detector thatdetects energy between 300 nm and 1700 nm. In one embodiment, siliconavalanche photodiodes can be used to detect wavelengths between 300 nmand 1100 nm. Indium gallium arsenic photodiodes can be used to detectwavelengths between 900 nm and 1700 nm. In some embodiments, an analyzersystem can comprise at least one detector; in other embodiments, theanalyzer system can comprise at least two detectors, and each detectorcan be chosen and configured to detect light energy at a specificwavelength range. For example, two separate detectors can be used todetect particles that have been tagged with different labels, which uponexcitation with an EM source, will emit photons with energy in differentspectra. In one embodiment, an analyzer system can comprise a firstdetector that can detect fluorescent energy in the range of 450-700 nmsuch as that emitted by a green dye (e.g. Alexa 546); and a seconddetector that can detect fluorescent energy in the range of 620-780 nmsuch as that emitted by a far-red dye (e.g. Alexa 647). Detectors fordetecting fluorescent energy in the range of 400-600 nm such as thatemitted by blue dyes (e.g. Hoechst 33342), and for detecting energy inthe range of 560-700 nm such as that emitted by red dyes (Alexa 546 andCy3) can also be used.

A system comprising two or more detectors can be used to detectindividual particles that are each tagged with two or more labels thatemit light in different spectra. For example, two different detectorscan detect an antibody that has been tagged with two different dyelabels. Alternatively, an analyzer system comprising two detectors canbe used to detect particles of different types, each type being taggedwith a different dye molecules, or with a mixture of two or more dyemolecules. For example, two different detectors can be used to detecttwo different types of antibodies that recognize two different proteins,each type being tagged with a different dye label or with a mixture oftwo or more dye label molecules. By varying the proportion of the two ormore dye label molecules, two or more different particle types can beindividually detected using two detectors. It is understood that threeor more detectors can be used without departing from the scope of theinvention.

It should be understood by one skilled in the art that one or moredetectors can be configured at each interrogation space, whether one ormore interrogation spaces are defined within a flow cell, and that eachdetector may be configured to detect any of the characteristics of theemitted electromagnetic radiation listed above. The use of multipledetectors, e.g., for multiple interrogation spaces, has been previouslydisclosed in a prior application and is incorporated by reference herefrom U.S. patent application Ser. No. 11/048,660. Once a particle islabeled to render it detectable (or if the particle possesses anintrinsic characteristic rendering it detectable), any suitabledetection mechanism known in the art may be used without departing fromthe scope of the present invention, for example a CCD camera, a videoinput module camera, a Streak camera, a bolometer, a photodiode, aphotodiode array, avalanche photodiodes, and photomultipliers producingsequential signals, and combinations thereof. Different characteristicsof the electromagnetic radiation may be detected including: emissionwavelength, emission intensity, burst size, burst duration, fluorescencepolarization, and any combination thereof.

C. Sampling System

In a further embodiment, the analyzer system may include a samplingsystem to prepare the sample for introduction into the analyzer system.The sampling system included is capable of automatically sampling aplurality of samples and providing a fluid communication between asample container and a first interrogation space.

In some embodiments, the analyzer system of the invention includes asampling system for introducing an aliquot of a sample into the singleparticle analyzer for analysis. Any mechanism that can introduce asample may be used Samples can be drawn up using either a vacuum suctioncreated by a pump or by pressure applied to the sample that would pushliquid into the tube, or by any other mechanism that serves to introducethe sample into the sampling tube. Generally, but not necessarily, thesampling system introduces a sample of known sample volume into thesingle particle analyzer; in some embodiments where the presence orabsence of a particle or particles is detected, precise knowledge of thesample size is not critical. In preferred embodiments the samplingsystem provides automated sampling for a single sample or a plurality ofsamples. In embodiments where a sample of known volume is introducedinto the system, the sampling system provides a sample for analysis ofmore than about 0.0001, 0.001, 0.01, 0.1, 1, 2, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, 500, 1000, 1500, or 2000 μl. In someembodiments the sampling system provides a sample for analysis of lessthan about 2000, 1000, 500, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20,10, 5, 2, 1, 0.1, 0.01, or 0.001 μl. In some embodiments the samplingsystem provides a sample for analysis of between about 0.01 and 1500 μl,or about 0.1 and 1000 μl, or about 1 and 500 μl, or about 1 and 100 μl,or about 1 and 50 μl, or about 1 and 20 μl. In some embodiments, thesampling system provides a sample for analysis between about 5 μl and200 μl, or about 5 μl and about 100 μl, or about 5 μl and 50 μl. In someembodiments, the sampling system provides a sample for analysis betweenabout 10 μl and 200 μl, or between about 10 μl and 100 μl, or betweenabout 10 μl and 50 μl. In some embodiments, the sampling system providesa sample for analysis between about 0.5 μl and about 50 μl.

In some embodiments, the sampling system provides a sample size that canbe varied from sample to sample. In these embodiments, the sample sizemay be any one of the sample sizes described herein, and may be changedwith every sample, or with sets of samples, as desired.

Sample volume accuracy, and sample to sample volume precision of thesampling system, is required for the analysis at hand. In someembodiments, the precision of the sampling volume is determined by thepumps used, typically represented by a CV of less than about 50, 40, 30,20, 10, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01% of sample volume. Insome embodiments, the sample to sample precision of the sampling systemis represented by a CV of less than about 50, 40, 30, 20, 10, 5, 4, 3,2, 1, 0.5, 0.1, 0.05, or 0.01%. In some embodiments, the intra-assayprecision of the sampling system is represented by a CV of less thanabout 10, 5, 1, 0.5, or 0.1%. In some embodiments, the intra-assayprecision of the sampling system shows a CV of less than about 5%. Insome embodiments, the interassay precision of the sampling system isrepresented by a CV of less than about 10, 5, or 1%. In someembodiments, the interassay precision of the sampling system shows a CVof less than about 5%.

In some embodiments, the sampling system provides low sample carryover,advantageous in that an additional wash step is not required betweensamples. Thus, in some embodiments, sample carryover is less than about1, 0.5, 0.1, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, or 0.001%. In someembodiments, sample carryover is less than about 0.02%. In someembodiments, sample carryover is less than about 0.01%.

In some embodiments the sampler provides a sample loop. In theseembodiments, multiple samples are drawn into tubing sequentially andeach is separated from the others by a “plug” of buffer. The samplestypically are read one after the other with no flushing in between.Flushing is done once at the end of the loop. In embodiments where abuffer “plug” is used, the plug may be recovered ejecting the bufferplug into a separate well of a microtiter plate.

The sampling system may be adapted for use with standard assayequipment, for example, a 96-well microtiter plate, or, preferably, a384-well plate. In some embodiments the system includes a 96 well platepositioner and a mechanism to dip the sample tube into and out of thewells, e.g., a mechanism providing movement along the X, Y, and Z axes.In some embodiments, the sampling system provides multiple samplingtubes from which samples may be stored and extracted from, when testingis commenced. In some embodiments, all samples from the multiple tubesare analyzed on one detector. In other embodiments, multiple singlemolecule detectors may be connected to the sample tubes Samples may beprepared by steps that include operations performed on sample in thewells of the plate prior to sampling by the sampling system, or samplemay be prepared within the analyzer system, or some combination of both.

D. Sample Preparation System

Sample preparation includes the steps necessary to prepare a raw samplefor analysis. These steps can involve, by way of example, one or moresteps of: separation steps such as centrifugation, filtration,distillation, chromatography; concentration, cell lysis, alteration ofpH, addition of buffer, addition of diluents, addition of reagents,heating or cooling, addition of label, binding of label, cross-linkingwith illumination, separation of unbound label, inactivation and/orremoval of interfering compounds and any other steps necessary for thesample to be prepared for analysis by the single particle analyzer. Insome embodiments, blood is treated to separate out plasma or serum.Additional labeling, removal of unbound label, and/or dilution steps mayalso be performed on the serum or plasma sample

In some embodiments, the analyzer system includes a sample preparationsystem that performs some or all of the processes needed to provide asample ready for analysis by the single particle analyzer. This systemmay perform any or all of the steps listed above for sample preparation.In some embodiments samples are partially processed by the samplepreparation system of the analyzer system. Thus, in some embodiments, asample may be partially processed outside the analyzer system first. Forexample, the sample may be centrifuged first. The sample may then bepartially processed inside the analyzer by a sample preparation system.Processing inside the analyzer includes labeling the sample, mixing thesample with a buffer and other processing steps that will be known toone in the art. In some embodiments, a blood sample is processed outsidethe analyzer system to provide a serum or plasma sample, which isintroduced into the analyzer system and further processed by a samplepreparation system to label the particle or particles of interest and,optionally, to remove unbound label. In other embodiments preparation ofthe sample can include immunodepletion of the sample to remove particlesthat are not of interest or to remove particles that can interfere withsample analysis. In yet other embodiments, the sample can be depleted ofparticles that can interfere with the analysis of the sample. Forexample, sample preparation can include the depletion of heterophilicantibodies, which are known to interfere with immunoassays that usenon-human antibodies to directly or indirectly detect a particle ofinterest. Similarly, other proteins that interfere with measurements ofthe particles of interest can be removed from the sample usingantibodies that recognize the interfering proteins.

In some embodiments, the sample can be subjected to solid phaseextraction prior to being assayed and analyzed. For example, a serumsample that is assayed for cAMP can first be subjected to solid phaseextraction using a c18 column to which it binds Other proteins such asproteases, lipases and phosphatases are washed from the column, and thecAMP is eluted essentially free of proteins that can degrade orinterfere with measurements of cAMP. Solid phase extraction can be usedto remove the basic matrix of a sample, which can diminish thesensitivity of the assay. In yet other embodiments, the particles ofinterest present in a sample may be concentrated by drying orlyophilizing a sample and solubilizing the particles in a smaller volumethan that of the original sample. For example, a sample of exhaledbreath condensate (EBC) can be dried and resuspended in a small volumeof a suitable buffer solution to enhance the detection of the particleof interest.

In some embodiments the analyzer system provides a sample preparationsystem that provides complete preparation of the sample to be analyzedon the system, such as complete preparation of a blood sample, a salivasample, a urine sample, a cerebrospinal fluid sample, a lymph sample, aBAL sample, an exhaled breath condensate sample (EBC), a biopsy sample,a forensic sample, a bioterrorism sample, and the like. In someembodiments the analyzer system provides a sample preparation systemthat provides some or all of the sample preparation. In someembodiments, the initial sample is a blood sample that is furtherprocessed by the analyzer system. In some embodiments, the sample is aserum or plasma sample that is further processed by the analyzer system.The serum or plasma sample may be further processed by, e.g., contactingwith a label that binds to a particle or particles of interest; thesample may then be used with or without removal of unbound label.

In some embodiments, sample preparation is performed, either outside theanalysis system or in the sample preparation component of the analysissystem, on one or more microtiter plates, such as a 96-well plate.Reservoirs of reagents, buffers, and the like can be in intermittentfluid communication with the wells of the plate by means of tubing orother appropriate structures, as are well-known in the art Samples maybe prepared separately in 96 well plates or tubes. Sample isolation,label binding and, if necessary, label separation steps may be done onone plate. In some embodiments, prepared particles are then releasedfrom the plate and samples are moved into tubes for sampling into thesample analysis system. In some embodiments, all steps of thepreparation of the sample are done on one plate and the analysis systemacquires sample directly from the plate. Although this embodiment isdescribed in terms of a 96-well plate, it will be appreciated that anyvessel for containing one or more samples and suitable for preparationof sample may be used. For example, standard microtiter plates of 384 or1536 wells may be used. More generally, in some embodiments, the samplepreparation system is capable of holding and preparing more than about5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, 1000, 5000,or 10,000 samples. In some embodiments, multiple samples may be sampledfor analysis in multiple analyzer systems. Thus, in some embodiments, 2samples, or more than about 2, 3, 4, 5, 7, 10, 15 20, 50, or 100 samplesare sampled from the sample preparation system and run in parallel onmultiple sample analyzer systems.

Microfluidics systems may also be used for sample preparation and assample preparation systems that are part of analyzer systems, especiallyfor samples suspected of containing concentrations of particles highenough that detection requires smaller samples. Principles andtechniques of microfluidic manipulation are known in the art. See, e.g.,U.S. Pat. Nos. 4,979,824; 5,770,029; 5,755,942; 5,746,901; 5,681,751;5,658,413; 5,653,939; 5,653,859; 5,645,702; 5,605,662; 5,571,410;5,543,838; 5,480,614, 5,716,825; 5,603,351; 5,858,195; 5,863,801;5,955,028; 5,989,402; 6,041,515; 6,071,478; 6,355,420; 6,495,104;6,386,219; 6,606,609; 6,802,342; 6,749,734; 6,623,613; 6,554,744;6,361,671; 6,143,152; 6,132,580; 5,274,240; 6,689,323; 6,783,992;6,537,437; 6,599,436; 6,811,668 and published PCT patent application no.WO9955461(A1) Samples may be prepared in series or in parallel, for usein a single or multiple analyzer systems.

Preferably, the sample comprises a buffer. The buffer may be mixed withthe sample outside the analyzer system, or it may be provided by thesample preparation mechanism. While any suitable buffer can be used, thepreferable buffer has low fluorescence background, is inert to thedetectably labeled particle, can maintain the working pH and, inembodiments wherein the motive force is electrokinetic, has suitableionic strength for electrophoresis. The buffer concentration can be anysuitable concentration, such as in the range from about 1 to about 200mM. Any buffer system may be used as long as it provides for solubility,function, and delectability of the molecules of interest. Preferably,for application using pumping, the buffer is selected from the groupconsisting of phosphate, glycine, acetate, citrate, acidulate,carbonate/bicarbonate, imidazole, triethanolamine, glycine amide,borate, MES, Bis-Tris, ADA, aces, PIPES, MOPSO, Bis-Tris Propane, BES,MOPS, IES, HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS,Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO,AMP, CAPS, and CABS. The buffer can also be selected from the groupconsisting of Gly-Gly, bicine, tricine, 2-morpholine ethanesulfonic acid(MES), 4-morpholine propanesulfonic acid (MOPS) and2-amino-2-methyl-1-propanol hydrochloride (AMP). A useful buffer is 2 mMTris/borate at pH 8.1, but Tris/glycine and Tris/HCl are alsoacceptable. Other buffers are as described herein.

Buffers useful for electrophoresis are disclosed in a prior applicationand are incorporated by reference herein from U.S. patent applicationSer. No. 11/048,660.

E. Sample Recovery

One highly useful feature of embodiments of the analyzers and analysissystems of the invention is that the sample can be analyzed withoutconsuming it. This can be especially important when sample materials arelimited. Recovering the sample also allows one to do other analyses orreanalyze it. The advantages of this feature for applications wheresample size is limited and/or where the ability to reanalyze the sampleis desirable, e.g., forensic, drug screening, and clinical diagnosticapplications, will be apparent to those of skill in the art.

Thus, in some embodiments, the analyzer system of the invention furtherprovides a sample recovery system for sample recovery after analysis. Inthese embodiments, the system includes mechanisms and methods by whichthe sample is drawn into the analyzer, analyzed and then returned, e.g.,by the same path, to the sample holder, e.g., the sample tube. Becauseno sample is destroyed and because it does not enter any of the valvesor other tubing, it remains uncontaminated. In addition, because all thematerials in the sample path are highly inert, e.g., PEEK, fused silica,or sapphire, there is little contamination from the sample path. The useof the stepper motor controlled pumps (particularly the analysis pump)allows precise control of the volumes drawn up and pushed back out. Thisallows complete or nearly complete recovery of the sample with little ifany dilution by the flush buffer. Thus, in some embodiments, more thanabout 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or99.9% of the sample is recovered after analysis. In some embodiments,the recovered sample is undiluted. In some embodiments, the recoveredsample is diluted less than about 1.5-fold, 1.4-fold, 1.3-fold,1.2-fold, 1.1-fold, 1.05-fold, 1.01-fold, 1.005-fold, or 1.001-fold.

For sampling and/or sample recovery, any mechanism for transporting aliquid sample from a sample vessel to the analyzer may be used. In someembodiments the inlet end of the analysis capillary has attached a shortlength of tubing, e.g., PEEK tubing that can be dipped into a samplecontainer, e.g. a test tube or sample well, or can be held above a wastecontainer. When flushing, to clean the previous sample from theapparatus, this tube is positioned above the waste container to catchthe flush waste. When drawing a sample in, the tube is put into thesample well or test tube. Typically the sample is drawn in quickly, andthen pushed out slowly while observing particles within the sample.Alternatively, in some embodiments, the sample is drawn in slowly duringat least part of the draw-in cycle; the sample may be analyzed whilebeing slowly drawn in. This can be followed by a quick return of thesample and a quick flush. In some embodiments, the sample may beanalyzed both on the inward (draw-in) and outward (pull out) cycle,which improves counting statistics, e.g., of small and dilute samples,as well as confirming results, and the like. If it is desired to savethe sample, it can be pushed back out into the same sample well it camefrom, or to another. If saving the sample is not desired, the tubing ispositioned over the waste container.

VI. Methods Using Highly Sensitive Analysis of Cardiac Troponin

The methods of the present invention make possible measurement ofcardiac troponin levels at concentrations far lower than previouslymeasured. Although cardiac troponin is an accepted marker for cardiacmuscle damage, its usefulness has been limited by the fact that, withcurrent methods of analysis, it is only detectable after considerabledamage to cardiac muscle has occurred, because of the lack ofsensitivity of current methods. The Joint European Society ofCardiology/American College of Cardiology committee for the Redefinitionof Myocardial Infarction has recommended that an increased concentrationof cardiac troponin be defined as a measurement exceeding the 99^(th)percentile of the distribution of cardiac troponin concentrations in thereference group, a very low threshold. A total imprecision (CV) at thisdecision limit of <10% is recommended. However, the analyticalimprecision obtained with presently available immunoassays for cardiactroponins is not uniform, mainly at the low concentration range. Inaddition, the assays that are currently available lack sufficientsensitivity for detecting troponin levels in nonclinical (normal)subjects, and a true baseline or a level of troponin defined in a normalpopulation, has not been defined. The analyzer systems of the inventionhave been shown to be able to consistently detect levels of cTnI atconcentrations of less than 10 pg/ml with a total imprecision of lessthan 10% (See Examples). Thus, the invention provides methods fordiagnosis, prognosis, or methods of treatment based on the highlysensitive detection of cardiac troponin in individuals.

In some embodiments, the invention provides a method for determining adiagnosis, prognosis, or method of treatment in an individual by i)determining a concentration of cardiac troponin in a sample ordetermining the concentrations of cardiac troponin in a series ofsamples from the individual, where the concentration is determined by acardiac troponin assay with a limit of detection for the cardiactroponin in said sample of less than about 50, 40, 30, 10, 5, 4,3,2 or 1pg/ml, e.g., less than about 20 pg/ml; and ii) determining a diagnosis,prognosis, or method of treatment in said individual, based on theconcentration in the sample, or on the concentrations in the series ofsamples. The method of determining the concentration of cardiac troponinincludes any suitable method with the requisite sensitivity, e.g., themethods descried herein. In some embodiments, the methods utilize amethod of determining a concentration of cardiac troponin in the samplewhere the method comprises detecting single molecules of troponin, orcomplexes or fragments thereof.

In some embodiments, the threshold concentration of troponin isdetermined by analyzing samples, e.g., blood, serum, or plasma samples,from an apparently healthy population for cardiac troponin, e.g.,cardiac troponin I, and determining the level at which 80, 90, 95, 96,97, 98, 99, 99.5, or 99.9% of the population fall below that level(concentration). This value is the threshold value. In some embodiments,the threshold value is set at the 99^(th) percentile. In someembodiments, the analyzing is performed using a method with a level ofdetection for the cardiac troponin of less than about 50, 20, 10, 5, or1 pg/ml, e.g., less than about 5 pg/ml.

In some embodiments, the invention provides a method for determining adiagnosis, prognosis, or method of treatment in an individual bycomparing a value for a concentration of cardiac troponin in a samplefrom the individual with a normal value or a range of normal values forcardiac troponin, where the normal vale or range of normal values isdetermined by a cardiac troponin assay with a limit of detection for thecardiac troponin in said sample of less than about 50, 40, 30, 10, 5,4,3,2 or 1 pg/ml, e.g., less than about 20 pg/ml; and ii) determining adiagnosis, prognosis, or method of treatment in said individual, basedon comparison.

In some embodiments, the cardiac troponin is cardiac troponin I orcardiac troponin T. In some embodiments, the cardiac troponin is cardiactroponin T. In some embodiments, the cardiac troponin is cardiactroponin I. The method may use total troponin, e.g., total cTnI, orcTnT, or total cTnI+cTnT, as described herein, in determining adiagnosis, prognosis, or method of treatment. In some embodiments, themethod may use the concentration of free, complexed, or fragments of thecardiac troponin, or a comparison of these (e.g., a ratio), to determinea diagnosis, prognosis, or method of treatment.

A. Samples

The sample or series of samples may be any suitable sample; in someembodiments, the sample(s) will be blood, serum, or plasma. In someembodiments, the sample or series of samples are serum samples. Theindividual may be an animal, e.g., mammal, e.g., human

A single sample may be taken, or a series of samples may be taken. If aseries of samples is taken, they may be taken at any suitable interval,e.g., intervals of minutes, hours, days, weeks, months, or years. In anacute clinical setting, typically a series of samples will be taken overthe course of hours and days, with the samples separated by a matter ofhours. When an individual is followed for longer periods, sampleintervals may be months or years. Diagnosis, prognosis, or method oftreatment may be determined from a single sample, or from one or more ofa series of samples, or from changes in the series of samples, e.g., anincrease in concentration at a certain rate may indicate a severecondition whereas increase at a slower rate or no increase may indicatea relatively benign or less serious condition. The rate of change may bemeasured over the course of hours, days, weeks, months, or years. Rateof change in a given individual may, in some cases, be more relevantthan an absolute value. In acute setting, an extremely rapid rate ofchange, e.g., a “spike”, can indicate an imminent, ongoing, or recentcardiac event. In other settings, a rise in values over a period ofdays, weeks, months or years in an individual can indicate ongoing andworsening cardiac damage, e.g., cardiac damage due to a cardiaccondition (e.g., cardiac hypertrophy or congestive heart failure) orcardiac damage due to a non-cardiac condition (e.g., toxicity from drugexposure).

In some embodiments, at least one sample is taken during or after acardiac stress test. E.g., a sample may be taken before the stress test,and one or more samples taken during the test. Deviations in cardiactroponin levels between the sample before the test and the sample(s)taken during the test can provide diagnostic or prognostic information,e.g., indicate the likelihood of coronary artery disease or otherpathology associated with the cardiac muscle. Other comparisons may bedone as well, such as comparisons of any of the samples to normal orthreshold levels, or determination of a rate of change in theconcentration of cardiac troponin in the samples, all of which may yielduseful information regarding cardiac and cardiovascular health, as wellas other conditions as described herein.

In some embodiments, at least one sample is taken at or near the timethe individual presents to a health professional with one or moresymptoms indicative of a condition that may involve cardiac damage.Settings in which an individual may present to a health careprofessional include, but are not limited to ambulatory, urgent care,critical care, intensive care, monitoring unit, inpatient, outpatient,physician office, medical clinic, emergency response setting, includingan ambulance, and health screening settings. In some embodiments, one ormore samples are taken from the individual and are assayed for cardiactroponin locally, i.e., at or near the setting at which the samples aretaken. For example, an individual who presents at a hospital may haveone or more samples taken that are assayed for cardiac troponin withinthe hospital. For example, an individual who presents at a hospital mayhave one or more samples taken that are assayed for cardiac troponinwithin the hospital. In some embodiments, one or more samples are takenfrom the individual and are assayed for cardiac troponin in a CLIAlaboratory. In some embodiments, the individual displays one or moresymptoms consistent with acute coronary syndrome. In some embodiments,the individual displays one or more symptoms consistent with AMI. Suchsymptoms include, but are not limited to, chest pain, chest pressure,arm pain, abnormal EKG, abnormal enzyme levels, and shortness of breath.

B. Determination of Diagnosis, Prognosis, or Method of Treatment

In some embodiments, step ii) includes comparing said concentration orseries of concentrations to a normal value for said concentration,comparing said concentration or series of concentrations to apredetermined threshold level, comparing said concentration or series ofconcentrations to a baseline value, or determining a rate of change ofconcentration for said series of concentrations.

In some embodiments, step ii) comprises comparing said concentration oftroponin in said sample with a predetermined threshold concentration,and determining a diagnosis, prognosis, or method of treatment if thesample concentration is greater than the threshold level. The thresholdconcentration can be determined by, e.g., determining the 99thpercentile concentration of troponin in a group of individuals, andsetting said threshold concentration at said 99th percentileconcentration. An example of this is given in Examples.

Normal values, threshold values, rates of change, ratios of values, andother useful diagnostic and prognostic indicators may be established bymethods well-known in the art. For example, these values may bedetermined by comparing samples from a case population and a controlpopulation, where the case population exhibits the biological state forwhich diagnosis, prognosis, or method of treatment is desired, and thecontrol population does not exhibit the biological state. In someembodiments, a longitudinal study may be done, e.g., the case populationmay be a subset of the control population that, over time, exhibits thebiological state. It will be appreciated that data from a plurality ofstudies may be used to determine a consensus value or range of valuesfor normal, and for prognostic or diagnostic levels.

In developing diagnostic or prognostic test, data for one or morepotential markers may be obtained from a group of subjects. The group ofsubjects is divided into at least two sets, and preferably the first setand the second set each have an approximately equal number of subjects.The first set includes subjects who have been confirmed as having adisease or, more generally, being in a first condition state. Forexample, this first set of patients may be those that have recently hada disease incidence, or may be those having a specific type of disease,such as AMI. The confirmation of the condition state may be made througha more rigorous and/or expensive testing such as MRI or CT. Hereinafter,subjects in this first set will be referred to as “diseased”. The secondset of subjects is simply those who do not fall within the first set.Subjects in this second set may be “non-diseased;” that is, normalsubjects. Alternatively, subjects in this second set may be selected toexhibit one symptom or a constellation of symptoms that mimic thosesymptoms exhibited by the “diseased” subjects. In still anotheralternative, this second set may represent those at a different timepoint from disease incidence. Preferably, data for the same set ofmarkers is available for each patient. This set of markers may includeall candidate markers which may be suspected as being relevant to thedetection of a particular disease or condition. Actual known relevanceis not required. Embodiments of the compositions, methods and systemsdescribed herein may be used to determine which of the candidate markersare most relevant to the diagnosis of the disease or condition. Thelevels of each marker in the two sets of subjects may be distributedacross a broad range, e.g., as a Gaussian distribution. However, nodistribution fit is required.

1. Acute Myocardial Infarct

The methods of the invention are especially useful in diagnosis,prognosis, and/or treatment selection in patients suspected of acutemyocardial infarct (AMI). Single or serial cardiac troponin measurementsin patients suspected of AMI provide incremental prognostic informationthat improves the prognosis and indicates appropriate and earlytherapeutic intervention to minimize the risk of adverse outcomes.

Thus, the invention provides a method of diagnosing, predicting, and/orpreventing or treating AMI in an individual by assaying a sample fromthe individual, e.g., a blood sample, plasma sample, and/or serumsample, for cardiac troponin, e.g., cTnI, and detecting a concentrationof cardiac troponin in the sample at a limit of detection of less thanabout 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pg/ml, e.g.,less than about 20 pg/ml, wherein the concentration of cardiac troponinin the sample indicates or predicts AMI. The cardiac troponin may becTnI or cTnT, and may be total troponin or a measure of a particularform, e.g., free, complexed, or fragment; in some embodiments, a ratioof one or more forms of the troponin is used, as described herein. Insome embodiments, total cTnI is measured in the sample or series ofsamples. In some embodiments, total cTnT is measured in the sample orseries of samples. In some embodiments, total cTnI+cTnT is measured inthe sample or series of samples. In some embodiments, the cardiactroponin level is determined at or near the time the individual presentsto a health professional with symptoms indicative of AMI. Such symptomsinclude, but are not limited to, chest pain, chest pressure, arm pain,abnormal EKG, abnormal enzyme levels, and shortness of breath.

In some embodiments, a series of measurements is taken, and a spike inthe cardiac troponin concentration in the samples indicates, predicts,or provides a basis for prognosis of AMI. In some embodiments, a spikeof over 50%, over 100%, over 150%, over 200%, over 250%, over 300%, over400%, or over 500% of baseline indicates, predicts, or provides a basisfor prognosis of AMI. In some embodiments, a cardiac troponin level ofover about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or50 pg/ml in a single sample indicates, predicts, or provides a basis forprognosis of AMI, regardless of baseline levels, if obtained. In someembodiments, a cardiac troponin level of about 1-10, or about 5-15, orabout 10-50, about 10-200, about 10-100, or about 10-40, or about 15-50,or about 15-40, or about 20-200, about 20-150, about 20-100, about20-50, or about 20-40, or about 20-30 pg/ml indicates, predicts, orprovides a basis for prognosis of AMI.

In some embodiments diagnosis or prognosis includes stratification forthe individual, based on cardiac troponin concentration in the sample orseries of samples. Such stratification may be based on the concentrationof cardiac troponin in single samples, presence of spikes and/or size ofspikes from baseline in a series of samples, ratios of different formsof the cardiac troponin, absolute values for different forms of cardiactroponin, rate of change in concentration for the cardiac troponin orfor one or more forms of the cardiac troponin in a series of samples,change in ratios of different forms of cardiac troponin over time in aseries of samples, and any other information based at least in part oncardiac troponin concentration in the sample or series of samples.Stratification may be based on values obtained from populations ofnormal and diseased subjects, as described herein. Appropriate treatmentmay also be determined based on the stratification of the individual.

In some embodiments, concentration of cardiac troponin is determined incombination with one or more other markers, e.g., markers of myocardialischemia, myocardial infarct or markers of stroke, and theconcentrations of each marker are considered in determining thediagnosis, prognosis, or method of treatment. Other clinical indicationstypically will also be taken into account, e.g., EKG, symptoms, history,and the like, as will be apparent to those of skill in the art.Appropriate algorithms for diagnosis, prognosis, or treatment may beconstructed based on the combinations of such markers and clinicalindications in combination with troponin levels.

Markers useful in combination with cardiac troponin in the methods ofthe invention include but are not limited to creatine kinase (CK) andits myocardial fraction CK myocardial band (MB), aspartateaminotransferase, lactate dehydrogenase (LDH), α-hydroxybutyratedehaydrogenase, myoglobin, glutamate oxaloacetate transaminase, glycogenphosphorylase BB, unbound free fatty acids, heart fatty acid bindingprotein (H-FABP), ischemia-modified albumin, myosin light chain 1,myosin light chain 2. Markers of inflammation and plaque instabilityuseful in combination with cardiac troponin in the methods of theinvention include but are not limited to C-reactive protein, white bloodcell count, soluble CD40 ligand, myeloperoxidase, monocytechemoattractant protein-1, whole blood choline, and pregnancy-associatedplasma protein A. Other markers of inflammation may be detected, andinclude combinations of 11-8, IL-1β, IL6, IL10, TNF, and IL-12p70, aswell as other cytokines or markers that will be apparent to those ofskill in the art.

In some embodiments, cardiac troponin, e.g., cTnI, is measured together,e.g., in the same sample, or in samples from the same individual takenat or near the same time, with a marker selected from the groupconsisting of creatine kinase (CK) and its myocardial fraction CKmyocardial band (MB), aspartate aminotransferase, lactate dehydrogenase(LDH), α-hydroxybutyrate dehaydrogenase, myoglobin, glutamateoxaloacetate transaminase, glycogen phosphorylase BB, unbound free fattyacids, heart fatty acid binding protein (H-FABP), ischemia-modifiedalbumin, myosin light chain 1, and myosin light chain 2 In someembodiments cardiac troponin, e.g., cTnI, is measured together withCK-MB, e.g., in the same sample, or in samples from the same individualtaken at or near the same time.

In some embodiments, cardiac troponin, alone or in combination withother markers or clinical signs, measured as described herein, is usedto determine reinfarction. In some embodiments, cardiac troponin, aloneor in combination with other markers or clinical signs, measured asdescribed herein, is used to determine characteristics of an infarct,e.g., size, or duration since infarct. In the latter case, fragments oftroponin produced by proteolysis in the blood may be compared to totaltroponin; the greater the proportion of fragments, the more time haselapsed since infarct.

2. Conditions Other than AMI

The methods of the invention also include methods of diagnosis,prognosis, and treatment based on concentration of cardiac troponin in asample that are useful in conditions other than AMI.

Many conditions include potential or actual cardiac damage, and theability to measure cardiac troponin at the levels described herein allowfor early detection of such damage and early intervention. Knowledge ofthe concentration of cardiac troponin as measured by the methods andcompositions of the invention is useful in diagnosis, prognosis, anddetermination of treatment for such conditions. Conditions includepercutaneous coronary interventions, cardiac surgery, heart failure,acute rheumatic fever, amyloidosis, cardiac trauma (including contusion,ablation, pacing, firing, cardioversion, catheterization and cardiacsurgery), reperfusion injury, cardiotoxicity from cancer therapy,congestive heart failure, end-stage renal failure, glycogen storagedisease type II (Pompe's disease), heart transplantation,haeomoglobinopathy with transfusion haemosiderosis, hypertension,including gestational hypertension, hypotension, often with arrhythmias,hypothyroidism, myocarditis, pericarditis, post-operative non-cardiacsurgery, pulmonary embolism, and sepsis.

In these embodiments, the troponin levels may be determinedconcomitantly with the levels of marker(s) that are specific for thenon-cardiac disease, or other symptoms or clinical signs of the disease;the marker(s) concentration and/or information regarding other symptomsor clinical signs is combined with information regarding cardiactroponin concentrations, determined as described herein, to determine adiagnosis, prognosis, and/or method of treatment. For example,embodiments of the invention may employ, in addition to determination ofcardiac troponin concentration, determination of the concentration ofone or more of the polypeptides referenced above, or other proteinmarkers useful in diagnosis, prognosis, or differentiation of disease.In some embodiments, a panel of markers for the disease is provided,where the panel includes cardiac troponin concentration, as describedherein, and at least on other marker for the disease. The panel mayinclude, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or individualmarkers, which including one or more cardiac troponins, e.g., totalcTnI. The analysis of a single marker or subsets of marker can becarried out by one skilled in the art to optimize clinical sensitivityor specificity in various clinical settings. These include, but are notlimited to ambulatory, urgent care, critical care, intensive care,monitoring unit, inpatient, outpatient, physician office, medicalclinic, and health screening settings. Furthermore, one skilled in theart can use a single marker or a subset of markers in combination withan adjustment of the diagnostic threshold in each of the aforementionedsettings to optimize clinical sensitivity and specificity.

a. Cardiac toxicity The compositions and methods of the invention areespecially useful in determining and monitoring cardiac toxicity thatresults from a treatment, e.g., cardiac toxicity of drug treatment.Thus, for example, the invention provides a method of assessing cardiactoxicity of a treatment by measuring cardiac troponin in an individualby i) determining a concentration of cardiac troponin in a sample ordetermining the concentrations of cardiac troponin in a series ofsamples from the individual, where at least one of the samples is takenfrom the individual during or after a time when the individual isreceiving the treatment, where the concentration or concentrations isdetermined by a cardiac troponin assay with a limit of detection for thecardiac troponin in said sample of less than about 50, 40, 30, 10, 5,4,3,2 or 1 pg/ml, e.g., less than about 20 pg/ml; and ii) assessing thedegree of cardiac toxicity of the treatment based on said concentrationor concentrations. In some embodiments, the treatment is a drugtreatment. In some embodiments, the treatment is a non-drug treatment.The method of determining the concentration of cardiac troponin includesany suitable method with the requisite sensitivity, e.g., the methodsdescribed herein. In some embodiments, the methods utilize a method ofdetermining a concentration of cardiac troponin in the sample where themethod comprises detecting single molecules of troponin, or complexes orfragments thereof.

Especially useful are methods of determination of cardiac toxicityutilizing the cross-reacting antibodies described herein, i.e.,antibodies that react with troponin from at least two species, such ashumans and another species such as rat, dog, mouse, or monkey. Suchantibodies may be used in animal studies of drug toxicity, where theindividual for which toxicity is assessed is, e.g., a mammal, such as arat, mouse, dog, monkey, or other animal used in such studies. Toxicityin various species may be directly compared when the antibody used inthe assay is the same antibody, thus reducing variability.

It will be appreciated that the compositions and methods of theinvention may be used in conjunction with specific drugs whose sideeffects include cardiotoxicity in order to monitor the cardiac toxicity.Thus, the invention provides methods of monitoring cardiac toxicity inan individual who is receiving a drug that is known to cause cardiactoxicity by determining the concentration of cardiac troponin in one ormore samples obtained from the individual, where the concentration orconcentrations is determined by a cardiac troponin assay with a limit ofdetection for the cardiac troponin in said sample or samples of lessthan about 50, 40, 30, 10, 5, 4,3,2 or 1 pg/ml, e.g., less than about 20pg/ml; and ii) assessing the degree of cardiac toxicity of the drugtreatment based on said concentration or concentrations. In someembodiments the method further includes a step iii) determining whetheror not to continue the drug treatment based on the assessment of stepii). Drugs whose side effects include cardiac toxicity are well-known inthe art.

C. Serial Testing

A clinical need for very high sensitivity troponin is developing andwith it the development of highly sensitive assays for the detection oftroponin as described herein. There are three major areas wherenext-generation troponin assays, e.g. assays for cTnI, can potentiallyimprove current practice. These ways include the earlier diagnosis of anindividual than is currently possible with existing routine markerse.g., with troponin, Creatine kinase-MB, and myoglobin, improved riskstratification for future adverse cardiac events among AMI ruleouts, andmonitoring of therapeutic drugs that have the potential to causecardiotoxicity. For AMI patients, the first detectable increase introponin after the onset of chest pain will enable an earlier diagnosisand triage of patients to the appropriate level of cardiac care. Thesensitivity of the analyzer, compositions, and assays described hereinhas the improved analytical sensitivity and precision to lower the 99thpercentile cutoff value. In particular the analyzer system can be usedto detect low concentrations of troponin levels and to detect thebiological variability (the variability between samples), referencechange values, and the index of individuality between samples. Thereference change value (RCV) is used to interpret serial results. Forserial results to be significantly different, the difference in thenumerical results must be greater than the combined variation inherentin the two results. The index of individuality corresponds to the ratioof the within subject biological variation to the between-subjectvariation. Increasing the analytical sensitivity will result in alowering of the cutoff concentration at the 99th percentile recommendedby. The Joint European Society of Cardiology/American College ofCardiology committee for the Redefinition of Myocardial Infarction, andwill result in a higher frequency of positive troponin results inpatients who present to the emergency room with chest pain. Unlessischemia can be ruled out by clinical or other means, a mildly increaseddetected level of troponin will produce more false positive cases ofAMI. In order to reduce the diagnostic confusion caused by highsensitivity troponin assays, the National Academy of ClinicalBiochemistry advocate the use of serial testing, using a more rigorousdetermination of statistically significant reference change values thatare based on the detection or measurement of an analyte's biologicvariation in a patient.

The determination of the biological variation for cardiac troponin hasnot been previously possible due to the lack of analytical sensitivityof assays to reliably measure troponin in the blood of healthy subjects.The availability of high sensitivity troponin assays now enables thereliable detection of troponin due to the normal turnover of cardiacmyocytes with the requisite imprecision (≦10%). The biological variationdata does not influence the interpretation of patients who have a veryhigh troponin concentration, e.g., a 10-fold increases relative to the99th percentile cutoff. These patients should be immediately admittedand treated without a need for serial troponin testing. In contrast,proper use of the biological variation will be important in theinterpretation of minor increases of troponin, i.e., values at or justabove the 99th percentile limit when very high sensitivity assays areused. Serial cardiac troponin values that exceed the upper RCV in theclinical context of chest pain increase the likelihood that the patientis suffering an evolving AMI. This interpretation may be valid even ifall serial troponin results are below the population-based referencelimit (due to the low index of individuality). However, there may beother acute disease processes that can increase troponin concentrationsover the short term, such as sepsis. Patients with serial troponinvalues that are mildly increased but are unchanging would increase thelikelihood that a chronic cardiac condition is present that is known tocause cardiac damage (e.g., myocarditis, heart and kidney failure).Patients with declining serial troponin results might indicate aresolving AMI, particularly if there is a history of chest pain from theprior days or week. It should be noted that patients with a positivetroponin due to a chronic disease can present with an acute exacerbationand increasing troponin values that mimic an AMI release pattern.Therefore clinical judgment remains paramount in the interpretation oftroponin testing.

Using the highly sensitive analyzers described previously, an individualhaving one or more symptoms of cardiac infarct can be classified with aseries of steps comprising: i) obtaining a sample from said individual,ii) detecting the level of troponin in the sample and iii) if the levelof troponin is above 10-fold of the 99th percentile, taking a firstaction with respect to the individual and if the level of troponin isbelow 10-fold of the 99th percentile taking a second action. In someembodiments, the first action is taken if patient has about a 3-fold toabout a 10-fold increase relative to the 99th percentile cutoff. In someembodiments, the first action is taken if patient has about a 4-fold toabout a 5-fold increase relative to the 99th percentile cutoff. In someembodiments, the first action is taken if patient has about a 6-fold toabout a 7-fold increase relative to the 99th percentile cutoff. In someembodiments, the first action is taken if patient has about an 8-fold toabout a 10-fold increase relative to the 99th percentile cutoff. In someembodiments, the first action is taken if patient has about a 1.5-foldto about a 2-fold increase. In some embodiments, the first action istaken if patient has about a 2-fold to about a 2.5-fold increase. Insome embodiments, the first action is taken if patient has about a2.5-fold to about a 3-fold increase. In some embodiments, the firstaction is taken if patient has about a 3-fold to about a 3.5-foldincrease. In some embodiments, the first action is taken if patient hasabout a 3.5-fold to about a 4-fold increase. In some embodiments, thefirst action is taken if patient has about a 4-fold to about a 4.5-foldincrease. In some embodiments, the first action is taken if patient hasabout a 4.5-fold to about a 5-fold increase. In some embodiments, thefirst action is taken if patient has about a 5-fold to about a 5.5-foldincrease. In some embodiments, the first action is taken if patient hasabout a 5.5-fold to about a 6-fold increase. In some embodiments, thefirst action is taken if patient has about a 6-fold to about a 6.5-foldincrease. In some embodiments, the first action is taken if patient hasabout a 6.5-fold to about a 7-fold increase. In some embodiments, thefirst action is taken if patient has about a 7-fold to about a 7.5-foldincrease. In some embodiments, the first action is taken if patient hasabout a 7.5-fold to about an 8-fold increase. In some embodiments, thefirst action is taken if patient has about a 8-fold to about a 8.5-foldincrease. In some embodiments, the first action is taken if patient hasabout an 8.5-fold to about a 9-fold increase. In some embodiments, thefirst action is taken if patient has about a 9-fold to about a 9.5-foldincrease. In some embodiments, the first action is taken if patient hasabout a 9.5-fold to about a 10-fold increase. In some embodiments thefirst action is taken if the increase is more than about 2-fold. In someembodiments the first action is taken if the increase is more than about3-fold. In some embodiments the first action is taken if the increase ismore than about 4-fold. In some embodiments the first action is taken ifthe increase is more than about 5-fold. In some embodiments the firstaction is taken if the increase is more than about 6-fold. In someembodiments the first action is taken if the increase is more than about7-fold. In some embodiments the first action is taken if the increase ismore than about 8-fold. In some embodiments the first action is taken ifthe increase is more than about 9-fold. In some embodiments the firstaction is taken if the increase is more than about 10-fold.

In some embodiments, the individual is a patient being evaluated for apossible cardiac event. In such a case, the first action can beadmission of the individual to a hospital, or other appropriate clinicalaction for treatment of the cardiac event. The second action can be tohold the individual for a period of time for further observation. Duringthe time the individual is held a series of samples are taken from saidpatient and the level of troponin in each sample detected. In someembodiments, the interval between samples is less than about 4, 3, 2, 1,0.5 hours apart. In some embodiments, the sample interval is less thanabout 4 hours apart. In some embodiments, the sample interval is about 1hour. In some embodiments, the sample interval is about 2 hours. It willbe recognized that circumstances in the clinic dictate sample intervalsand some degree of variation is acceptable and is encompassed within theinvention, as long as results may be reliably interpreted. In someembodiments, in addition to the level of troponin detected in eachsample, the rate of change in the level of troponin over two or moresamples in the series of samples is detected. In some embodiments, thechange in the level of troponin is a decrease. In some embodiments, thechange in the level of troponin is an increase. A decision can be maderegarding a course of action for said individual based on said rate ofchange, e.g., if the rate of change in troponin values exceeds apredetermined upper reference rate of change value, the decision is toadmit the individual. It will be recognized that other clinical factorswell-known to those of skill in the art can be used to modify thedecision based on troponin levels, e.g., if other clinicalmanifestations of acute myocardial infarct are present, such as abnormalEKG, abnormal enzyme levels, physical symptoms, and the like, then thethreshold for troponin that leads to an admission is suitably decreased.

The decision to take a first action, e.g., to admit a patient, can alsobe made if one or more spikes in the level of troponin are seen fromsample to sample. The spike can be any increase in the level of troponindetected compared to one or more samples wherein the increase iscompared to a threshold level. For example, if the threshold level isdetermined to be 10 pg/ml and if in one of the sample of the series ofsamples the troponin level detected is 15 pg/ml, the patient isadmitted. In some embodiments, the threshold level is determined from areference population. In some embodiments, the threshold level is set ata level for an individual, e.g., the initial level of troponin detectedfrom the first sample taken from the patient. In these embodiments, arelative increase indicates a spike, no matter what the absolute valuesare. In some embodiments, a spike is determined when the troponin levelincreases 2, 3, 4, 5, 6, 7, 8, 9 10, or more than 10-fold above areference value for the individual, e.g., a value set at the troponinlevel in the first sample taken from the individual. Alternatively, aspike in the level of troponin can be seen when comparing troponinlevels between samples from the individual. In such a case, no matterwhat the value of the level of troponin detected, the patient isadmitted if the value increases by more than a certain amount, e.g., apatient is admitted when the level of troponin increases by 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10 pg/ml as compared to one or moreneighboring samples. In some embodiments, the change in the level oftroponin indicates an acute cardiovascular disease. In some embodiments,the change in the level of troponin indicates a chronic cardiovasculardisease. In some embodiments, the change in the level of troponinindicates cardiotoxicity.

The existing commercial assays lack the sensitivity to low to detectvery minor and chronic increases. With the development of high sensitiveassays, the utility of cardiac troponin should be re-examined for thisapplication. If proven to be useful, the long-term biological variationand calculation of the RCV will be relevant.

D. Business Methods

The present invention relates to systems and methods (including businessmethods) for establishing markers of cardiac troponin that can be usedfor diagnosing, prognosing, or determining a method of treatment of abiological state or a condition in an organism, preparing diagnosticsbased on such markers, and commercializing/marketing diagnostics andservices utilizing such diagnostics. The biological state may be acutemyocardial infarct, or cardiac damage due to drug toxicity, or non-AMIstates as described herein.

In one embodiment, the business methods herein comprise: establishingone or more cardiac troponin markers using a method comprising:establishing a range of concentrations for said marker or markers inbiological samples obtained from a first population by measuring theconcentrations of the marker or markers in the biological samples bydetecting single molecules of the marker or markers at a level ofdetection of less than about 50, 20, 10, 5, or 1 pg/ml; andcommercializing the one or more markers established in the above step,e.g., in a diagnostic product. The diagnostic product herein can includeone or more antibodies that specifically binds to the cardiac troponinmarker and a fluorescent moiety that is capable of emitting an averageof at least about 200 photons when simulated by a laser emitting lightat the excitation wavelength of the moiety, where the laser is focusedon a spot of not less than about 5 microns in diameter that contains themoiety, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules.

In one embodiment, the business methods herein comprise: establishing arange of normal values for a cardiac troponin marker using a systemcomprising: establishing a range of concentrations for said cardiactroponin marker in biological samples obtained from a first populationby measuring the concentrations of the marker the biological samples bydetecting single molecules of the marker at a level of detection lessthan about 50, 20, 10, 5, or 1 pg/ml; and providing a diagnostic serviceto determine if an organism has or does not have a state or condition ofinterest, e.g., AMI, cardiac toxicity due to drug treatment, or anon-AMI condition. A diagnostic service herein may be provided by a CLIAapproved laboratory that is licensed under the business or the businessitself. The diagnostic services herein can be provided directly to ahealth care provider, a health care insurer, or a patient. Thus thebusiness methods herein can make revenue from selling e.g., diagnosticservices or diagnostic products.

The business methods herein also contemplate providing diagnosticservices to, for example, health care providers, insurers, patients,etc. The business herein can provide diagnostic services by eithercontracting out with a service lab or setting up a service lab (underClinical Laboratory Improvement Amendment (CLIA) or other regulatoryapproval). Such service lab can then carry out the methods disclosedherein to identify if a cardiac troponin marker is within a sample.

VII. Compositions

The invention provides compositions useful in the detection andquantitation of cardiac troponin. Compositions include binding partnersto cardiac troponin that are labeled with suitable labels for detectionby the methods of the invention, pairs of binding partners in which oneor both of the binding partners are labeled with suitable labels fordetection by the methods of the invention, solid supports to whichcapture binding partners are attached, in some embodiments also withdetection binding partners.

Exemplary embodiments include a composition for the detection of cardiactroponin that includes a binding partner to the cardiac troponinattached to a fluorescent moiety, where the fluorescent moiety iscapable of emitting an average of at least about 200 photons whensimulated by a laser emitting light at the excitation wavelength of themoiety, where the laser is focused on a spot of not less than about 5microns in diameter that contains the moiety, and wherein the totalenergy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the binding partner includes anantibody to the cardiac troponin. In some embodiments, the antibody is apolyclonal antibody. In some embodiments, the antibody is a monoclonalantibody. In some embodiments, the antibody is a cross-reactingantibody, e.g., an antibody that cross-reacts with cardiac troponin fromat least two species, e.g., at least two species selected from the groupconsisting of human, monkey, dog, and mouse. In some embodiments theantibody cross-reacts with cardiac troponins from all of human, monkey,dog, and mouse. In some embodiments, the cardiac troponin is selectedfrom the group consisting of cTnI and cTnT. In some embodiments, thecardiac troponin is cTnI. In some embodiments, cardiac troponin is cTnT.The antibody may specific to a specific region of the troponin molecule,e.g., specific to a region comprising amino acids 27-41 of cardiactroponin I. The fluorescent moiety may contain one or more moleculesthat comprises at least one substituted indolium ring system in whichthe substituent on the 3-carbon of the indolium ring contains achemically reactive group or a conjugated group. The label compositionmay include a fluorescent moiety that includes one or more dye moleculesselected from the group consisting of ALEXA-FLUOR® 488, 532, 647, 700,or 750. The label composition may include a fluorescent moiety thatincludes one or more dye molecules selected from the group consisting ofALEXA-FLUOR® 488, 532, 700, or 750. The label composition may include afluorescent moiety that includes one or more dye molecules that areALEXA-FLUOR® 488. The label composition may include a fluorescent moietythat includes one or more dye molecules that are ALEXA-FLUOR® 555. Thelabel composition may include a fluorescent moiety that includes one ormore dye molecules that are ALEXA-FLUOR® 610. The label composition mayinclude a fluorescent moiety that includes one or more dye moleculesthat are ALEXA-FLUOR® 647. The label composition may include afluorescent moiety that includes one or more dye molecules that areALEXA-FLUOR® 680. The label composition may include a fluorescent moietythat includes one or more dye molecules that are ALEXA-FLUOR® 700. Thelabel composition may include a fluorescent moiety that includes one ormore dye molecules that are ALEXA-FLUOR® 750.

In some embodiments, the invention provides a composition that includesa set of standards for the determination of a concentration of a cardiactroponin, wherein at least one of the standards is at a concentration ofcardiac troponin less than about 20, 15, 10, 5, 4, 3, 2, or 1 pg/ml. Insome embodiments, the invention provides a composition that includes aset of standards for the determination of a concentration of a cardiactroponin, wherein at least one of the standards is at a concentration ofcardiac troponin less than about 20 pg/ml. In some embodiments, theinvention provides a composition that includes a set of standards forthe determination of a concentration of a cardiac troponin, wherein atleast one of the standards is at a concentration of cardiac troponinless than about 10 pg/ml. In some embodiments, the invention provides acomposition that includes a set of standards for the determination of aconcentration of a cardiac troponin, wherein at least one of thestandards is at a concentration of cardiac troponin less than about 5pg/ml. In some embodiments, the invention provides a composition thatincludes a set of standards for the determination of a concentration ofa cardiac troponin, wherein at least one of the standards is at aconcentration of cardiac troponin less than about 1 pg/ml.

Other compositions of the invention are as described herein.

VIII. Kits

The invention further provides kits. Kits of the invention include oneor more compositions useful for the sensitive detection of cardiactroponin, as described herein, in suitable packaging. In someembodiments kits of the invention provide labels, e.g., binding partnersuch as an antibody that is specific for cardiac troponin, where thebinding partner is attached to a fluorescent moiety. In some embodimentskits of the invention provide binding partner pairs, e.g., antibodypairs, that are specific for cardiac troponin, where at least one of thebinding partners is a label for a cardiac troponin, as described herein.In some embodiments, the binding partners, e.g., antibodies, areprovided in separate containers. In some embodiments, the bindingpartners, e.g., antibodies, are provided in the same container. In someembodiments, one of the binding partners, e.g., antibody, is immobilizedon a solid support, e.g., a microtiter plate or a paramagnetic bead. Insome of these embodiments, the other binding partner, e.g., antibody, islabeled with a fluorescent moiety as described herein.

Binding partners, e.g., antibodies, solid supports, and fluorescentlabels for components of the kits may be any suitable such components asdescribed herein.

The kits may additionally include reagents useful in the methods of theinvention, e.g., buffers and other reagents used in binding reactions,washes, buffers or other reagents for preconditioning the instrument onwhich assays will be run, and elution buffers or other reagents forrunning samples through the instrument.

Kits may include one or more standards, e.g., standards for use in theassays of the invention, such as standards of highly purified, e.g.,recombinant, human cTnI or human cTnT, or various fragments, complexes,and the like, thereof. Kits may further include instructions.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limiting the remaining disclosure.

Unless otherwise specified, processing samples in the Examples wereanalyzed in a single molecule detector (SMD) as described herein, withthe following parameters: Laser: continuous wave gallium arsenite diodelaser of wavelength 639 nm (Blue Sky Research, Milpitas, Calif.),focused to a spot size of approximately 2 microns (interrogation spaceof 0.004 pL as defined herein); flow rate=5 microliter/min through afused silica capillary of 100 micron square ID and 300 micron square OD;non-confocal arrangement of lenses (see, e.g., FIG. 1A); focusing lensof 0.8 numerical aperture (Olympus); silicon avalanche photodiodedetector (Perkin Elmer, Waltham, Mass.).

Example 1 Sandwich Assays for Biomarkers: Cardiac Troponin I (cTnI)

The assay: The purpose of this assay was to detect the presence ofcardiac Troponin I (cTnI) in human serum. The assay format was atwo-step sandwich immunoassay based on a mouse monoclonal captureantibody and a goat polyclonal detection antibody. Ten microliters ofsample were required. The working range of the assay is 0-900 pg/ml witha typical analytical limit of detection of 1-3 pg/ml. The assay requiredabout four hours of bench time to complete.

Materials: the following materials were used in the procedure describedbelow: Assay plate: Nunc Maxisorp, product 464718, 384 well, clear,passively coated with monoclonal antibody, BiosPacific A34440228P Lot #A0316 (5 μg/ml in 0.05 M sodium carbonate pH 9.6, overnight at roomtemperature); blocked with 5% sucrose, 1% BSA in PBS, and stored at 4oC.For the standard curve, Human cardiac Troponin I (BiosPacific Cat #J34000352) was used. The diluent for the standard concentrations washuman serum that was immonodepleted of endogenous cTnI, aliquoted andstored at−20oC. Dilution of the standards was done in a 96 well,conical, polypropylene, (Nunc product #249944). The following buffersand solutions were used: (a) assay buffer: BBS with 1% BSA and 0.1%TritonX-100; (b) passive blocking solution in assay buffer containing 2mg/ml mouse IgG, (Equitech Bio); 2 mg/ml goat IgG, (Equitech Bio); and 2mg/ml MAK33 poly, Roche#11939661; (c) detection Antibody (Ab): GoatPolyclonal antibody affinity purified to Peptide 3, (BiosPacific G129C),which was label with a fluorescent dye ALEXA-FLUOR® 647, and stored at4oC; detection antibody diluent: 50% assay buffer, 50% passive blockingsolution; wash buffer: borate buffer saline Triton Buffer (BBST) (1.0 Mborate, 15.0 M sodium chloride, 10% Triton X-100, pH 8.3); elutionbuffer: BBS with 4M urea, 0.02% Triton X-100 and 0.001% BSA.

Preparation of ALEXA-FLUOR® 647 labeled antibodies: the detectionantibody G-129-C was conjugated to ALEXA-FLUOR® 647 by first dissolving100 ug of G-129-C in 400 uL of the coupling buffer (0.1M NaHCO3). Theantibody solution was then concentrated to 50 ul by transferring thesolution into YM-30 filter and subjecting the solution and filter tocentrifugation. The YM-30 filter and antibody was then washed threetimes by adding 400 ul of the coupling buffer. The antibody wasrecovered by adding 50 □l to the filter, inverting the filter, andcentrifuging for 1 minute at 5,000×g. The resulting antibody solutionwas 1-2 ug/ul. ALEXA-FLUOR® 647 NHS ester was reconstituted by adding 20ul DMSO to one vial of ALEXA-FLUOR® 647, this solution was stored at−20oC for up to one month. 3 ul of ALEXA-FLUOR® 647 stock solution wasadded to the antibody solution, which was then mixed and incubated inthe dark for one hour. After the one hour, 7.5 ul 1M tris was added tothe antibody ALEXA-FLUOR® 647 solution and mixed. The solution wasultrafiltered with YM-30 to remove low molecular weight components. Thevolume of the retentate, which contained the antibody conjugated toALEXA-FLUOR® 647, was adjusted to 200-400 □l by adding PBS. 3 ul 10%NaN3 was added to the solution, the resulting solution was transferredto an Ultrafree 0.22 centrifugal unit and spun for 2 minutes at12,000×g. The filtrate containing the conjugated antibody was collectedand used in the assays.

Procedure: cTnI standard and sample preparation and analysis:

The standard curve was prepared as follows: working standards wereprepared (0-900 pg/ml) by serial dilutions of the stock of cTnI intostandard diluent or to achieve a range of cTnI concentrations of between1.2 pg/ml-4.3 μg/ml.

10 □l passive blocking solution and 10 μl of standard or of sample wereadded to each well. Standards were run in quadruplicate. The plate wassealed with Axyseal sealing film, centrifuged for 1 min at 3000 RPM, andincubated for 2 hours at 25° C. with shaking. The plate was washed fivetimes, and centrifuged until rotor reached 3000 RPM in an invertedposition over a paper towel. A 1 nM working dilution of detectionantibody was prepared, and 20 μl detection antibody were added to eachwell. The plate was sealed and centrifuged, and the assay incubated for1 hour at 25° C. with shaking. 30 μl elution buffer were added per well,the plate was sealed and the assay incubated for ½ hour at 25° C. Theplate was either stored for up to 48 hours at 4° C. prior to analysis,or the sample was analyzed immediately.

For analysis, 20 μl per well were acquired at 40 μl/minute, and 5 μlwere analyzed at 5 μl/minute. The data were analyzed based on athreshold of 4 sigma Raw signal versus concentration of the standardswas plotted. A linear fit was performed for the low concentration range,and a non-linear fit was performed for the full standard curve. Thelimit of detection (LoD) was calculated as LOD=(3×standard deviation ofzeros)/slope of linear fit. The concentrations of the samples weredetermined from the equation (linear or non-linear) appropriate for thesample signal

An aliquot was pumped into the analyzer. Individually-labeled antibodieswere measured during capillary flow by setting the interrogation volumesuch that the emission of only 1 fluorescent label was detected in adefined space following laser excitation. With each signal representinga digital event, this configuration enables extremely high analyticalsensitivities. Total fluorescent signal is determined as a sum of theindividual digital events. Each molecule counted is a positive datapoint with hundreds to thousands of DMC events/sample. The limit ofdetection the cTnI assay of the invention was determined by the mean+3SD method.

Results: Data for a typical cTnI standard curve measured inquadruplicate using the assay protocol is shown in Table 2.

TABLE 2 Standard Curve for cTnI cTnI Standard (pg/ml) Signal Deviation %CV 0 233 25 10.8 1.5625 346 31 8.9 3.125 463 35 7.5 6.25 695 39 5.6 12.51137 61 5.3 25 1988 139 7.0 50 3654 174 4.8 100 5493 350 6.4 200 8264267 3.2 400 9702 149 1.5 800 9976 50 0.5

The sensitivity of the analyzer system was tested in 15 runs and wasfound routinely to detect sub femtomol/l (fM) levels of calibrator, asshown by the data in Table 3. The precision was 10% at 4 and 12 pg/mlcTnI.

TABLE 3 Instrument Sensitivity Calibrator Signal (fM) counts CV 0 11 12302 9 60 1341 8 300 4784 7

Linearized standard curve for the range concentrations of cTnI are shownin FIG. 5.

The analytical limit of detection (LoD) was determined across 15sequential assays. The LoD was the mean of the 0 std+3 SD (n=4)intra-assay determinations. The average LoD was 1.7 pg/ml (range 0.4-2.8pg/ml).

The recovery of the sample was determined by analyzing samples of serumthat had been immunodepleted of cTnI and spiked with known amounts ofcTnI. Table 4 shows the data for sample recovery by the system analyzedover 3 days.

TABLE 4 Sample Recovery Spike Recovery Standard % (pg/ml) (mean)Deviation CV 5 5.7 0.9 16 15 13.7 0.2 2 45 43 0.6 2 135 151 6.2 4

The linearity of the assay was determined in pooled human serum that wasspiked with cTnI and diluted with standard diluent. The results in Table5 show the dilutions and % of the signal expected for the correspondingdilution.

TABLE 5 Assay Linearity Serum % of Dilution expected 1:2 79 1:4 87 1:896

These data show that the analyzer system of the invention allows forperforming highly sensitive laser-induced immunoassay for sub-femtomolarconcentrations of cTnI.

Example 2 Sandwich Bead-based Assays for TnI

The assays described above use the same microtiter plate format wherethe plastic surface is used to immobilize target molecules. The singleparticle analyzer system also is compatible with assays done in solutionusing microparticles or beads to achieve separation of bound fromunbound entities.

Materials: MyOne Streptavidin C1 microparticles (MPs) are obtained fromDynal (650.01-03, 10 mg/ml stock). Buffers use in the assay include: 10×borate buffer saline Triton Buffer (BBST) (1.0 M borate, 15.0 M sodiumchloride, 10% Triton X-100, pH 8.3); assay buffer (2 mg/ml normal goatIgG, 2 mg/ml normal mouse IgG, and 0.2 mg/ml MAB-33-IgG-Polymer in 0.1 MTris (pH 8.1), 0.025 M EDTA, 0.15 M NaCl, 0.1% BSA, 0.1% Triton X-100,and 0.1% NaN3, stored at 4 C); and elution buffer (BBS with 4 M urea,0.02% Triton X-100, and 0.001% BSA, stored at 2-8 C). Antibodies used inthe sandwich bead-based assay include: Bio-Ab (A34650228P (BiosPacific)with 1-2 biotins per IgG) and Det-Ab (G-129-C(BiosPacific) conjugated toA647, 2-4 fluors per IgG). The standard is recombinant human cardiactroponin I (BiosPacific, cat # J34120352). The calibrator diluent is 30mg/ml BSA in TBS wEDTA.

Microparticles Coating: 100 ul of the MPs stock is placed in aneppendorf tube. The MPs are washed three times with 100 ul of BB ST washbuffer by applying a magnet, removing the supernatant, removing themagnet, and resuspending in wash buffer. After the washes the MPs areresuspended in 100 ul of assay buffer and 15 ug of Bio-Ab are added. Themixture is then incubated for an hour at room temperature with constantmixing. The MPs are washed five times with 1 ml wash buffer as describedabove. After the washes the MPs are resuspended in 15 ml of assay buffer(or 100 ul to store at 4 0C).

Preparation of Standard and Samples: The standard is diluted withcalibrator diluent to prepare proper standard curve (usually 200 pg/mldown to 0.1 pg/ml). Frozen serum and plasma samples need to becentrifuged 10 minutes at room temperature at 13K rpm. Clarifiedserum/plasma is removed carefully to avoid taking any possible pelletsor floaters and put into fresh tubes. 50 ul of each standard or sampleis pipetted into appropriate wells.

Capture Target: 150 ul of MPs (after resuspension to 15 ml in assaybuffer+400 mM NaCl) are added to each well. The mixture is incubated onJitterBug, 5 at room temperature for 1 hr.

Washes and Detection: The plate is placed on a magnet and thesupernatant is removed after ensuring that all MPs are captured by themagnet. 250 ul of wash buffer are added after removing the plate fromthe magnet. The plate is then placed on the magnet and the supernatantis removed after ensuring that all MPs are captured by the magnet. 20 ulDet-Ab are added per well (Det-Ab to 500 ng/ml is diluted in assaybuffer+400 mM NaCl)). The mixture is incubated on JitterBug, 5 at roomtemperature for 30 min

Washes and Elution: The plate is placed on a magnet and washed threetimes with wash buffer. The supernatant is removed after ensuring thatall MPs are captured by the magnet and 250 ul of wash buffer are added.After the washes the samples are transferred into a new 96-well plate.The new plate is then placed on the magnet and the supernatant isremoved after ensuring that all MPs are captured by the magnet. 250 ulof wash buffer are then added after removing the plate from the magnet.The plate is then placed on the magnet and the supernatant is removedafter ensuring that all MPs are captured by the magnet. 20 ul of elutionbuffer are then added and the mixture is incubated on JitterBug, 5 atroom temperature for 30 min

Filter out MPs and transfer to 384-well plate: The standard and samplesare transferred into a 384-well filter plate placed on top of a 384-wellassay plate. The plate is then centrifuged at room temperature at 3000rpm with a plate rotor. The filter plate is removed and the appropriatecalibrators are added. The plate is covered and is ready to be run onSMD.

SMD: An aliquot is pumped into the analyzer. Individually-labeledantibodies are measured during capillary flow by setting theinterrogation volume such that the emission of only 1 fluorescentmolecule is detected in a defined space following laser excitation. Witheach signal representing a digital event, this configuration enablesextremely high analytical sensitivities. Total fluorescent signal isdetermined as a sum of the individual digital events. Each moleculecounted is a positive data point with hundreds to thousands of DMCevents/sample. The limit of detection the cTnI assay of the invention isdetermined by the mean+3 SD method.

Example 3 Concentration Range for cTnI in a Population of NormalNon-diseased Subjects

A reference range or normal range for cTnI concentrations in human serumwas established using serum samples from 88 apparently healthy subjects(non-diseased). A sandwich immunoassay as described in Example 1 wasperformed and the number of signals or events as described above werecounted using the single particle analyzer system of the invention. Theconcentration of serum troponin I was determined by correlating thesignals detected by the analyzer with the standard curve as describedabove. All assays were perfumed in quadruplicate.

In accordance with recommendations by the current European and AmericanCardiology Societies (ESC/ACC) troponin assays should quantifyaccurately the 99th percentile of the normal range with an assayimprecision (CV) of less than 10% in order to distinguish reliablybetween patients with ACS and patients without ischemic heart disease,and risk stratification for adverse cardiac events. The assay showedthat the biological threshold (cutoff concentration) for TnI is at a TnIconcentration of 7 pg/ml, which is established at the 99th percentilewith a corresponding CV of 10% (FIG. 5). At the 10% CV level theprecision profile points at a TnI concentration of 4 and 12 pg/ml.

In addition, the assay correlates well with the Troponin-I standardmeasurements provided by the National Institute of Standards andTechnology (FIG. 6).

The assay of the invention is sufficiently sensitive and precise tofulfill the requirements of the ESC/ACC, and it is the most sensitiveassay for cardiac troponin I when compared to assays such as thosedescribed by Koerbin et al. (Ann Clin Biochem, 42:19-23 (2005). Theassay of the invention has a 10-20 fold greater sensitivity than thatcurrently available assays, which has determined the biologicalthreshold range to be 111-333 pg/ml cTnI.

Example 4 Detection of Early Release of TnI into the Circulation ofPatients with Acute Myocardial Infarction (AMI)

Study 1: 47 samples were obtained serially from 18 patients thatpresented with chest pain in the emergency department (ED). Thesepatients all had non-ST elevated ECG were, and were diagnosed with AMI.The concentration of cTnI in the initial samples from all 18 patientswas determined according to a commercial assay at the time of admissionto the emergency room to be <350 pg/ml (10% cutpoint), and 12 were <100pg/ml (99th %) percentile. These samples were tested at later timesusing the same commercial assay, and were determined to test positivefor cTnI. The same serum samples were also assayed for TnI according tothe assay of the invention as described in Examples 1 and 3, and theresults compared to the results obtained using the commercial assay.

Blood was drawn for the first time at the time the patient presentedwith chest pain (sample 1), and subsequently at intervals between 4-8hours (samples 2 at 12 hours; sample 3 at 16 hours; sample 4 at 24hours; sample 5 at 30 hours; sample 6 at 36 hours; sample 7 at 42 hours;and sample 8 at 48 hours). The serum was analyzed by the methods of theinvention and by a current commercial method, and the results obtainedare shown in FIG. 7. The analyzer of the invention detected TnI at thetime the patient presented with chest pain (sample 1), while thecommercial assay first detected cTnI at a much later time (sample 6 at36 hours). The concentration of TnI in sample 3 exceeded the biologicalthreshold level that was established using the analyzer of the invention(7 pg/ml, see FIG. 5), and indicated that sample 3 is positive for TnIto suggest the incidence of a cardiac event. The biological thresholdfor the commercial assay lies between 111 and 333 pg/ml of TnI.Accordingly, sample 3 would not have been considered to indicate apossible cardiac event.

In addition, the methods and compositions of the present invention allowfor much earlier diagnosis and possible intervention based on cardiactroponin levels, as evidenced by results for the first sample taken fromthe patients. In the 3 cases that had initial commercial assay cTnIvalues of between 100 and 350 ng/ml, all were positive for cTnI by theanalytical methods of the invention (i.e., cTnI over 7 pg/ml). In the 12cases that had initial commercial cTnI values of less than 100 pg/ml, 5were determined to be positive for a cardiovascular event according tothe assay of the invention (i.e., cTnI over 7 pg/ml). The prospectiveuse of the assay of the invention would have detected 53% more AMI casesthan the current commercial assay when the admission sample was tested.

Study 2: 50 additional serum samples, which tested negative according tothe commercial assay, were tested using the analyzer and assay of theinvention. The results are shown in FIG. 8. Of the 50 samples, 36 werewithin the 99th % and determined to be within the normal rangeestablished by the assay of the invention. However, the remaining 14samples that were determined to be within the commercial “normal” ornon-diseased range, tested above the biological threshold established bythe invention.

Therefore, the high sensitivity cTnI assay of the invention allows forthe detection of myocardial damage in patients when cTnI serum levelsare below threshold values by commercially available technology. The useof the highly sensitive and precise cTnI assay of the invention enablesdetection of AMI earlier than with existing cTnI assays, and therebyprovides the opportunity for appropriate diagnosis and early medicalintervention to improve the outcome.

Example 5 Sandwich Bead-based Assays for TnI

The single particle analyzer system is compatible with assays done insolution using microparticles or beads to achieve separation of boundfrom unbound entities, e.g., in the analysis of cardiac troponin I.

Materials: MyOne Streptavidin C1 microparticles (MPs) are obtained fromDynal (650.01-03, 10 mg/ml stock). Buffers use in the assay include: 10×borate buffer saline Triton Buffer (BBST) (1.0 M borate, 15.0 M sodiumchloride, 10% Triton X-100, pH 8.3); assay buffer (2 mg/ml normal goatIgG, 2 mg/ml normal mouse IgG, and 0.2 mg/ml MAB-33-IgG-Polymer in 0.1 MTris (pH 8.1), 0.025 M EDTA, 0.15 M NaCl, 0.1% BSA, 0.1% Triton X-100,and 0.1% NaN3, stored at 4 C); and elution buffer (BBS with 4 M urea,0.02% Triton X-100, and 0.001% BSA, stored at 2-8 C). Antibodies used inthe sandwich bead-based assay include: Bio-Ab (A34650228P (BiosPacific)with 1-2 biotins per IgG) and Det-Ab (G-129-C(BiosPacific) conjugated toA647, 2-4 fluors per IgG). The standard is recombinant human cardiactroponin I (BiosPacific, cat # J34120352). The calibrator diluent is 30mg/ml BSA in TBS wEDTA.

Microparticles Coating: 100 ul of the MPs stock is placed in aneppendorf tube. The MPs are washed three times with 100 ul of BB ST washbuffer by applying a magnet, removing the supernatant, removing themagnet, and resuspending in wash buffer. After the washes the MPs areresuspended in 100 ul of assay buffer and 15 ug of Bio-Ab are added. Themixture is then incubated for an hour at room temperature with constantmixing. The MPs are washed five times with 1 ml wash buffer as describedabove. After the washes the MPs are resuspended in 15 ml of assay buffer(or 100 ul to store at 40C).

Preparation of Standard and Samples: The standard is diluted withcalibrator diluent to prepare proper standard curve (usually 200 pg/mldown to 0.1 pg/ml). Frozen serum and plasma samples need to becentrifuged 10 minutes at room temperature at 13K rpm. Clarifiedserum/plasma is removed carefully to avoid taking any possible pelletsor floaters and put into fresh tubes. 50 ul of each standard or sampleis pipetted into appropriate wells.

Capture Target: 150 ul of MPs (after resuspension to 15 ml in assaybuffer+400 mM NaCl) are added to each well. The mixture is incubated onJitterBug, 5 at room temperature for 1 hr.

Washes and Detection: The plate is placed on a magnet and thesupernatant is removed after ensuring that all MPs are captured by themagnet. 250 ul of wash buffer are added after removing the plate fromthe magnet. The plate is then placed on the magnet and the supernatantis removed after ensuring that all MPs are captured by the magnet. 20 ulDet-Ab are added per well (Det-Ab to 500 ng/ml is diluted in assaybuffer+400 mM NaCl)). The mixture is incubated on JitterBug, 5 at roomtemperature for 30 min

Washes and Elution: The plate is placed on a magnet and washed threetimes with wash buffer. The supernatant is removed after ensuring thatall MPs are captured by the magnet and 250 ul of wash buffer are added.After the washes the samples are transferred into a new 96-well plate.The new plate is then placed on the magnet and the supernatant isremoved after ensuring that all MPs are captured by the magnet. 250 ulof wash buffer are then added after removing the plate from the magnet.The plate is then placed on the magnet and the supernatant is removedafter ensuring that all MPs are captured by the magnet. 20 ul of elutionbuffer are then added and the mixture is incubated on JitterBug, 5 atroom temperature for 30 min

Filter out MPs and transfer to 384-well plate: The standard and samplesare transferred into a 384-well filter plate placed on top of a 384-wellassay plate. The plate is then centrifuged at room temperature at 3000rpm with a plate rotor. The filter plate is removed and the appropriatecalibrators are added. The plate is covered and is ready to be run onSMD.

SMD: An aliquot is pumped into the analyzer. Individually-labeledantibodies are measured during capillary flow by setting theinterrogation volume such that the emission of only 1 fluorescentmolecule is detected in a defined space following laser excitation. Witheach signal representing a digital event, this configuration enablesextremely high analytical sensitivities. Total fluorescent signal isdetermined as a sum of the individual digital events. Each moleculecounted is a positive data point with hundreds to thousands of DMCevents/sample. The limit of detection the cTnI assay of the invention isdetermined by the mean+3 SD method.

Example 6 Cross-reacting Antibodies

Cross-reacting antibody pairs were prepared for cross-reactivity withhuman, monkey, dog, and rat. Assays were performed on serum samplesusing a protocol essentially as described in Example 5, i.e., captureantibody was immobilized on paramagnetic beads. Results are shown inFIGS. 9 and 10. This Example demonstrates that antibodies thatcross-react to several species may be produced and used to measurecardiac troponin I at very low limits of detection, i.e., less than 1pg/ml across human, monkey, dog, and rat.

Example 7 Accurate and Reproducible IA-based Technology

This example describes a IA-based technology that can accurately andreproducibly measure proteins with a sensitivity of 10-100 femtogram(fg)/ml with a dynamic range of >4.5 logs in sample volumes of 50-150ul.

Materials and Methods

Materials

Antibodies and analytes (recombinant) were obtained from R&D Systems(Minneapolis, Minn.) Fluorescent dyes and biotin succimidyl ester, usedto label antibodies, were obtained from Invirtogen (San Diego, Calif.).Rat, dog, and monkey cTnI were purified from natural sources andobtained from Hytest (Sweden). Human lithium citrate plasma specimenswere purchased from Interstate blood bank Streptavidin coatedparamagnetic microparticles (MPs) were obtained from Invitrogen.Antibodies were labeled with fluorescent dye (detection antibody,usually polyclonal) and biotin (capture antibody, usually monoclonal)using manufacturers recommendations. MPs were coated with biotinylatedantibody under saturation conditions, washed and stored in assay buffer(1% BSA based buffer containing non-ionic detergent and heterophile/HAMAantibody blocking reagents).

Immunoassays

Unless stated otherwise, the typical immunoassay was performed asfollows. Samples or standards (e.g. 50 ul-100 ul) were diluted withassay buffer containing capture antibody coated MPs (e.g. 150 ul) andincubated in a 96 well plate for one to two hours at 25° C. withshaking. MPs were separated using a magnetic bed (Ambion, Tex.).Supernatant was removed and then 20 ul of detection antibody was addedand incubated for 60 minutes at 25° C. with shaking. The MPs were againmagnetically separated and washed six times using buffered saline plussurfactant After removal of residual wash buffer, 20 ul of elutionbuffer was added. This reagent disrupted antibody-analyte interactionsand resulted in the release of detection antibody from the MPs. Thesolution in each 96-well was then transferred to a 384-well filter plateand centrifuged at 3,000 RPM for three minutes to separate detectionantibody in elution buffer from MPs. The eluted and filtered material inthe 384-well plate was then placed into the analyzer system.

Analyzer System

The analyzer system is based upon single molecule counting technology.Liquid is sipped well and pumped through a capillary with a 100micro-meter diameter. The liquid passes through a interrogation spacecomprising a laser for fluorescent dye excitation and fluorescentemission is detected via confocal microscopy. The 4.5 plus log dynamicreporting range was obtained by using a combination of output signals.At low analyte concentrations, individual fluorescently-tagged detectionantibody molecules were counted as they passed through the interrogationspace in the instrument's flow cell. This signal is a sum of all suchmeasured events over a set time interval and is termed detected events(DE). At higher concentrations of analyte, multiple molecules weredetected simultaneously in the flow, and signal was measured as photonevents (PE) over the time interval. At this point, total light measuredduring the time event was used. At the highest concentrations ofanalyte, total photons (TP), the sum of all photon events, was used. DE,PE and TP signal were used to generate a weighted four-paramater curvefit for each signal type Finally, a computer algorithm was created thatcombined these three curve fits into a weighted single curve fit thatprovided a >4.5 log linear reporting range.

Results

Reporting Range, Linearity, Accuracy and Reproducibility

To determine the reporting range for the IL-6 assay, recombinant humanIL-6 was serially diluted (1 ng/ml to 10 fg/ml) in a goat serum-baseddiluent. Samples of 100 ul were run in quadruplicate. The reportingrange of a typical bioassay is presented in FIG. 11. Resulting signalwas back interpolated using the curve fit algorithm. A linear response(R²=0.99; y=0.97+6.4) was observed from 10 fg/ml to 400 pg/ml,representing an approximate 4.5 log reporting range (FIG. 11). Accuracywas determined by measuring spike recovery of analyte (IL-6 and humancardiac troponin-I, cTnI) into panels of human plasma. The IL-6 and cTnIassays and spike levels of 5 and 50 pg/ml, average % recoveries werebetween 90% and 110% for both analytes at both concentrations (data notshown). Results from performing the cTnI immunoassay over eightconsecutive runs and back interpolating the standard curves arepresented in FIG. 12. In these experiments, the highest concentrationstandard used was 100 pg/ml. A linear (R²=0.99) response was observed atboth the high and low ends of the standard curve shown. The % CV for the8 assay runs for back interpolated determinations was <10% between forall values >0.78 pg/ml. The % CV was 16% and 23% for the 0.39 and 0.2pg/ml values, respectively.

Sensitivity

Sensitivity or LoD for the analyzer, composition, and methods of theimmunoassay system varied from analyte to analyte. Table 6 depicts theLoD for 10 different human immunoassays. Larger sample volumesconsistently resulted in lower LoDs and enhanced sensitivity. Forexample, sample volumes >50 ul resulted in LoDs ranging from 0.01 pg/ml(human IL-6) to 0.12 pg/ml (human VEGF). As sample volumes decreased,LoD's increased in a proportional manner. All of the assays wereperformed in a two-step manner with 1-2 hours of capture and 1 hour ofdetection.

TABLE 6 The effect of sample volume on immunoassay sensitivity (LoD)Volume LoD Analyte (ul) LoD pg/ml Volume (ul) pg/ml MCP-1 200 0.03 200.25 RANTES 100 0.05 20 0.3 VEGF 100 0.12 20 1.0 IL-8 50 0.12 20 0.3IL-1a 200 0.01 20 0.1 IL-7 200 0.02 10 0.3 IL-6 100 0.01 10 0.15 TNF-a200 0.02 10 0.4 IL-1B 150 0.02 10 0.3 cTPN-I 100 0.11 10 1.2

The effect of incubation time on assay sensitivity is shown in Table 7.In this case a single step cTnI assay was performed using 50 ul ofsample and simultaneous capture and detection reactions. As incubationtimes, increased assay sensitivity improved. This was due to animprovement in slope response as a function of time. Of note, even witha 15 minute incubation a LoD of 0.61 pg/ml was achieved (using the NISTreference material).

TABLE 7 The effect of incubation time on a one step cTnI immunoassaysensitivity (LoD) Time (minutes) Background Slope LoD pg/ml 120 184 1520.241 60 172 102 0.336 30 173 90 0.383 15 193 64 0.61

To assess species cross reactivity, antibodies were chosen for the cTnIbioassay that should cross react across these species based upon cTnIhomology at reactive epitopes. Serial dilutions of individual cTnIs fromhuman (NIST material), rat, dog and monkey were made in a BSA-baseddiluent Sample volumes of 50 ul were then tested with the analyzersystem. The findings are presented in Table 8. Slopes of the dilutioncurves ranged from 68-138, and LoDs ranged 0.14-0.72 pg/ml. Bias againstthe human NIST standard was 1.6, 2.0, and 4.9 for rat, dog and monkey,respectively. The reason for this bias is not clear but may reflectinconsistencies in analyte value assignment by the manufacturer.Regardless all 4 species cTnI provided immunoassays with LoDs <1 pg/ml.

TABLE 8 Species cross reactivity of the analyzer system cTnIimmunoassay. SPECIES OF CTNI BACKGROUND SLOPE LOD (PG/ML) Human (NIST)143 138 0.19 Monkey 147 28 0.72 Dog 138 68 0.58 Rat 138 84 0.14

The sensitivity of the human cTnI assay was employed to define the rangeof cTnI in human plasma obtained from 100 human blood donors (50 ulsample size). Thee results are presented in FIG. 13. The values rangedfrom <2 pg/ml to 39 pg/ml with an average value of 2.19 pg/ml (4.1SD).Three plasma had values <0.2 pg/ml which was the LoD. The value for the99^(th) percentile was 9 pg/ml. If the one apparent outlier of 39 pg/mlwas removed from the data set the mean for the population was 1.83 pg/ml(1.9SD) with the 99^(th) percentile at 8 pg/ml.

This example shows the combination of ultra-sensitive flow detectionwith MP based IA technology provides protein bioassays with analyticalLODs in the fg/ml range, which is approximately 10-fold more sensitivethan previously demonstrated. We have applied this technology to createimportant bioassays for clinically useful protein biomarkers, such ascTnI and IL-6. This new development in IA technology provides accurate,sensitive and reproducible quantitative bioassays with a dynamic rangeof measurement in small sample volumes across multiple mammalianspecies.

The analyzer, compositions, and methods provide enhanced sensitivity viatwo mechanisms First, unlike analog systems that count total light (e.g.a colorimetric 96-well plate reader), the analyzer system separatesbackground fluorescent signal from antibody-tagged signal bythresholding out the background signal and measuring eachfluorescently-labeled detection antibody as a digital event (Wu et al).Second, the MP solid phase has been designed to minimize non-specificbinding (NSB) of fluorescently-labeled detection antibodies.Furthermore, incubations are performed in 96-well plates withpolypropylene wells that have very low protein binding capacity and,thus, low NSB. With the analyzer system, when a fluorescently-taggeddetection antibody is released from the MPs, the level of NSB inreactions that contain no analyte is very low. These characteristicshave allowed us to use relatively high concentrations of detectionantibody (100-500 ng/ml) which favor a steep assay response whilemaintaining a very low background.

The sensitivity of the analyzer, compositions, and methods allows forthe measurement and quantification of biomarkers in normal, non-diseasestates. This is an important foundation that must be established beforechanges in biomarkers can be used to diagnose disease and monitorprogression and therapeutic interventions. We quantified the levels ofcTnI in a panel of lithium heparin plasma obtained from 100 human blooddonors. The mean and range of cTnI values in this plasma are similar tothose reported previously for serum specimens. In the case of cTnI, wedemonstrated an analytical LoD of 0.2 pg/ml and a 10% CV cutpointbetween 0.78 and 1.6 pg/ml. Since normal subjects have serum and plasmacTnI levels between 0.3-9 pg/ml (average 2-3 pg/ml), increases fromthese normal levels can be measured with a high level of precision inmost human serum and plasma samples. We have also shown that theanalyzer, compositions, and methods provide analytical sensitivities <1pg/ml across multiple mammalian species that are often used inpreclinical studies, such as monkey, rat and dog. This finding may havesignificant applications in drug development where the monitoring ofcardiac toxicity in preclinical animal models is desired.

The enhanced sensitivity that analyzer, compositions, and methodsprovide has additional benefits; namely sample volume requirements andincubation times. For example, to achieve highest levels of sensitivity,often sample volume is increased in IA applications. By providing a moresome sensitive approach, less sample volume can be used with theanalyzer system thus enabling the preservation of precious specimens.One of the limitations of rapid tests used today near patient settingsis a lack of sensitivity compared to automated system counterparts. Thisis attributable to technology, sample volume, as well as incubationtimes. We demonstrated a proportional increase in sensitivity withincubation times. The analyzer, compositions, and methods havesensitivities of 10-100 fg/ml using 100 ul of sample and one to twohours incubation. Using a one-step modification for the analyzer systemas described above we were able to reduce the incubation times to 15minutes with an approximate LoD of 0.6 pg/ml with 50 ul of sample. Thisis 50-200 times more sensitive than a variety of commercially availablecTnI assays on automated platforms. Taken together these datademonstrate the potential for the development of a clinically usefulrapid sub pg/ml IA using single molecule counting as incorporated in theanalyzer system

Approaches to enhance ELISA sensitivity have involved the use of avariety of technologies including signal amplification (e.g. doubleenzymatic amplification). Although high sensitivity IAs have beendemonstrated using this approach, often the magnitude of assay dynamicrange is compromised to a 1.5-2.0 log range. If the analyte of interestexists in a wide biological range, such as for cytokines, then a samplewill often require numerous dilutions to achieve the correctconcentration for measurement in the IA.

The analyzer system builds single molecule detection combined with MP IAtechnology to provide highly sensitive immunoassays. It also provides abroad dynamic reporting range and flexible sample volume usage. Dynamicor broad reporting range is important in the ability to detect bothnormal and disease states in one standardized bioassay. The flexibilityof sample volume usage can be used to determine multiple quantificationsfrom a small sample or to conserve samples. If ultimate sensitivity isnot required, for example if the analyte is in the 20-100 pg/ml range,multiple analyses can be made. In the case where the normal range of aprotein biomarker is in the low 10-20 pg/ml range, a highly sensitiveassay could be combined with small sample requirements to enable sampleconservation and potential usage for the measurement of additionalbiomarkers. This could be especially important when analyzing rodentsamples or human repository specimens.

Example 8 Short and Long-Term Biological Variation for Cardiac TroponinI Using a High Sensitivity Assay: Implications for Clinical Practice

As serial testing will be important in the interpretation of lowconcentrations, we determined the short-term (4-hours) and long-term(8-weeks) biological variability, reference change values and index ofindividuality for cTnI. Assays for cardiac troponin have been themainstay for diagnosis of acute myocardial infarction (AMI) and riskstratification for future adverse cardiac events. In the clinicalcontext of myocardial ischemic, the European Society/American College ofCardiology have redefined AMI to be predicated on an increase in cardiactroponin above the 99^(th) percentile of a healthy population with aimprecision of 10% or less. Up until recently, there have been very fewcommercial assays that can detect cardiac troponin I (cTnI)concentrations in healthy subjects with the requisite precision.Recently, a new high sensitivity troponin assay was developed usingsingle photon fluorescence detection. In preliminary studies, this assayhad a sensitivity of 0.2 ng/L, and a 10% coefficient of variance (CV) at1.8 ng/L. The 99^(th) percentile cutoff concentration obtained from ahealthy population was lower at 10 ng/L than any of the other commercialassays for cTnI.

The clinical need for very high sensitivity troponin has been recentlyreviewed. There are three major areas where next-generation cTnI assayscan potentially improve current practice: earlier diagnosis than iscurrently possible with existing routine markers (troponin, Creatinekinase-MB, and myoglobin), improved risk stratification for futureadverse cardiac events among AMI ruleouts, and monitoring of therapeuticdrugs that have the potential to cause cardiotoxicity. For AMI patients,the first detectable increase in troponin after the onset of chest painwill enable an earlier diagnosis and triage of patients to theappropriate level of cardiac care. Melanson et al. showed that use of ahigh-sensitivity cTnI assay was positive earlier than use of a lowersensitivity assay 64% of confirmed AMI cases. For risk stratification,use of the lowest troponin cutoff (i.e., 99^(th) percentile) in theTIMI-TACTICS II trial identified 12% more cases of patients who weresubsequently determined to be at high risk for future AMI and cardiacdeaths. For cardiotoxicity, chemotherapeutic drugs such as theanthracyclines release small amounts of troponin as the result of injuryto the heart. Use of a high-sensitivity troponin assay may enabledetection of other drugs that are cardiotoxicity (e.g., cyclooxygenase-2inhibitors), where existing assays lack the requisite analyticalsensitivity.

Increasing the analytical sensitivity will result in a lowering of thecutoff concentration at the 99^(th) percentile, and will result in ahigher frequency of positive troponin results in patients who present tothe emergency department with chest pain. Unless ischemia can be ruledout by clinical or other means, a mildly increased troponin will producemore false positive cases of AMI. In order to reduce the diagnosticconfusion caused by high sensitivity troponin assays, the NationalAcademy of Clinical Biochemistry advocated the use of serial testing.Troponin concentrations that are stable over an appropriate samplinginterval are more likely caused by chronic diseases such as renalfailure, heart failure, sepsis, and myocarditis. The NACB recommended achange of 20% from baseline as suggestive of an AMI that is eitherevolving (rise in troponin) or resolving (fall in troponin). This limitwas determined based a calculation of three times the imprecision at thecutoff concentrations and not from the marker's biological variation.There have been no recommendations to date as to how serial results oftroponin should be interpreted in the context of risk stratification forfuture adverse events or cardiac toxicity due to therapeutic drugs.

A more rigorous determination of statistically significant referencechange values of serial testing is based on measurement of an analyte'sbiologic variation. These studies are usually conducted in healthysubjects with no evidence of active cardiovascular disease. However,until assays were available that could reliably detect troponin inhealthy subjects, such studies were not possible. Biological variabilitystudies cannot be conducted on diseased patients due to the changingconcentrations of troponin over time in patients with acute coronarysyndromes (ACS) or chronic diseases such as heart and renal failure.Therefore, we determined the short- and long-term biological variabilityon healthy subjects using a prototype cTnI assays with 10-50 timeshigher analytical sensitivity than the best that is available among thecommercially-available assays.

Materials and Methods

Patients and Specimens

Two separate cohorts of subjects were recruited for this study using aprotocol that was reviewed and approved by the University of CaliforniaCommittee for Human Research and the San Francisco General Hospital(SFGH) General Clinical Research Center (GCRC). All subjects signed awritten consent. For the short-term biological variability, 12 subjects(6 males and 6 females, age range 23-54 y) were recruited. A heparinlock was inserted into the antecubital vein by staff at the SFGH GCRC.Thereafter, blood was collected at 0, 1, 2, 3, and 4 hours through theheparin lock into vacutainer tubes containing no anticoagulant. A smallamount of blood was discarded prior to blood collection to flush theline of heparin. After collection, a small amount of heparin wasinjected into the intravenous lines to maintain patency. The collectedblood was allowed to clot, the tubes centrifuged within 30 minutes, andthe serum aliquoted and frozen at −70° C. until analysis. For thelong-term biological variability, 17 subjects (9 males and 10 females,age range 19-58 y) were recruited. Blood was collected throughvenipuncture every other week for 6-8 weeks (3-4 total samples persubject). Samples were centrifuged and stored at −70° C. and thawed(once) prior to analysis. To minimize the pre-analytical variability,the same phlebotomist was used for each subject. For the long-termstudy, blood was collected on the same day of the week and roughly thesame time of the day. For both cohorts, the subjects self-reported nosymptoms or history of heart disease.

Data Analysis

Frozen samples were allowed to thaw at room temperature. All analyticalassays for cardiac troponin were performed at Singulex Inc., Hayward,Calif. using the analyzer, compositions, and methods, and were tested induplicate using the same technologist on the same day (different daysfor each of the short- and long-term studies). All results andcalculations were performed using Microsoft Excel (Redmond, Wash.). Weuse the Cochran's test to determine the presence of outliers amongpatient results. We found that the distribution of troponin data wasnon-parametric and therefore performed a log normal transformation ofthe troponin results prior to calculation of the biological variation.The equations established by Fokkema et al. were used instead of themore widely-used nested ANOVA procedures of Fraser et al., who do notdescribe log transformation. For calculation of the reference changevalue, this transformation resulted in nonsymmetric limits, i.e.,separate criteria for statistically significant difference betweenincreasing and decreasing troponin values. We determined the goals forprecision (≦0.5×CV_(I)) and accuracy (≦0.25×(CV_(I) ²+CV_(T) ²)^(1/2))using the convention of Fraser et al.

In summary, for the assessment of short-term variation, blood wascollected on every hour for 4 hours (5 samples total each) on 12 healthysubjects. For the assessment of long-term variation, blood was collectedevery other week for 8 weeks (3-4 samples each) on 17 healthy subjects.Blood was tested using a high sensitivity cTnI assay (limit of detection0.2 ng/L). The analytical coefficient of variance (CV_(A)),intra-individual (CV_(I)) and total median (CV_(T)) variation, index ofindividuality were computed. As the data was non-parametricallydistributed, the reference change values (RCV) were calculated using alognormal approach

Results

The distribution of results for the short and long-term biologicalvariation for cardiac troponin is shown in FIGS. 14A and 14B,respectively. The mean (points) and range (bars) of values are given.The data shown in FIG. 14A is the short-term biological variation on 12subjects over 4 hours. The data shown in FIG. 14B is the long-termvariation on 15 subjects over 8 weeks. In both FIGS. 14A and 14B,statistical outliers were removed. There were no outliers from thehour-to-hour dataset. From the week-to-week dataset, we determined onesubject was determined to be an outlier and was removed. None of thepoints had values that were below the detection limit of the assay of0.2 ng/L, and all were below the 99^(th) percentile limit of 10 ng/L,previously determined on 100 apparently healthy subjects. Table 9 showsthe results of the short and long-term biological variability forcardiac troponin.

TABLE 9 Short and long-term biological variability (BV) for cTnIPARAMETER SHORT-TERM BV^(A) LONG-TERM BV^(B) Analytical variationCV_(A), %7.6 12.2 Biological variation CV_(I), % 7.8 9.0 CV_(T), % 10.915.2 Index of individuality 0.72 0.59 Precision goals, % 3.9 4.5Inaccuracy goals, % 3.4 5.2 RCV: lognormal increase 35 52 RCV: lognormaldecrease −26 −34 ^(A)Over 4 hours. ^(B)Over 8 weeks.The values CV_(I), and CV_(T) were slightly lower for the short-termrelative to the long-term. This is expected given that changes to theheart are not expected to change in a healthy subject from hour to hour,but may change slightly from week to week. Goals for imprecision,inaccuracy, and index of individuality are also shown with the resultsof the hour-to-hour values being slightly higher but not significantlydifferent from the week-to-week results.

The distribution of the hour-to-hour and day-to-day cTnI results werenon-parametric and right skewed and a lognormal transformation wasperformed prior to the calculation of RCV. This resulted in theproduction of non-symmetrical RCV results, with higher limits signifyingstatistical change for increasing cTnI results and lower limits fordecreasing results. The higher RCV limit for a rising troponin reflectsthe right skewed distribution of results from healthy subjects and thegreater statistical uncertainty when values are increasing. Thecalculated RCV results are about 1.5 to 2 times higher than the singlelimit of 20% change in serial results that was recommended by the NACB.This is partly due to the higher imprecision observed at the lower cTnIconcentrations observed for healthy subjects.

The short-term CV_(A), CV_(I) and CV_(T) for cTnI were 7.6% (mean 2.3ng/L), 7.8%, and 10.9%. Corresponding results for the long-termvariation were: 12.2%, 9.0%, and 15.2%. The index of individuality were0.72 and 0.59, respectively. The RCV were 35% (increase) and −26%(decrease) for short-term, and 52% and −34%, respectively with 95%confidence. The low index of individuality indicates that apopulation-based reference intervals are less useful than measuringserial change values in individual patients. This is particularlyimportant for the interpretation of patients who have low concentrationincreases in cTnI using very high-sensitivity assays for patientspresenting with chest pain (short-term) and for evaluation of drugs forcardiotoxicity (long-term).

The determination of the biological variation for cardiac troponin hasnot been previously possible due to the lack of analytical sensitivityof assays to reliably measure troponin in the blood of healthy subjects.The availability of high sensitivity troponin assays now enables thereliable detection of troponin due to the normal turnover of cardiacmyocytes with the requisite imprecision (≦10%). Two different researchassays have now shown that there is a non-parametric distribution ofcardiac troponin in healthy subjects. The results in this study wereconsistent with these previous findings.

Table 10 compares the biological variation for cardiac troponin whenmeasured over the short term, against the other commonly used cardiacbiomarkers of ACS. Total CK, CK-MB activity, CK-MB mass, and myoglobinare described as “event markers,” such as plaque erosion in the case ofAMI. The CV_(I) for these troponin and CK-MK mass are low relative tothe CV_(T), producing a low index of individuality. An low index (<0.6)indicates that reference intervals will be of little utility forinterpretation of individual results whereas an index that his high(>1.4) indicates that reference values are of significant value. Table10 shows that the traditional ACS markers have a low index. In thisstudy, the short-term troponin index was slightly higher at 0.72 thanthe other markers, reinforcing the notion that serial testing is animportant criteria for diagnosis of low level increases.

The biological variation data does not influence the interpretation ofpatients who have a very high troponin concentration, e.g., a 10-foldincreases relative to the 99^(th) percentile cutoff. These patientsshould be immediately admitted and treated without a need for serialtroponin testing. In contrast, proper use of the biological variationwill be important in the interpretation of minor increases of troponin,i.e., values at or just above the 99^(th) percentile limit when veryhigh sensitivity assays are used. Serial cardiac troponin values thatexceed the upper RCV in the clinical context of chest pain increase thelikelihood that the patient is suffering an evolving AMI. Thisinterpretation may be valid even if all serial troponin results arebelow the population-based reference limit (due to the low index ofindividuality). However, there may be other acute disease processes thatcan increase troponin concentrations over the short term, such assepsis. Patients with serial troponin values that are mildly increasedbut are unchanging would increase the likelihood that a chronic cardiaccondition is present that is known to cause cardiac damage (e.g.,myocarditis, heart and kidney failure). Patients with declining serialtroponin results might indicate a resolving AMI, particularly if thereis a history of chest pain from the prior days or week. It should benoted that patients with a positive troponin due to a chronic diseasecan present with an acute exacerbation and increasing troponin valuesthat mimic an AMI release pattern. Therefore clinical judgment remainsparamount in the interpretation of troponin testing.

Table 10 also compares long-term biological variation of cardiactroponin against other biomarkers that are used for chronic diseasedetection.

TABLE 10 Summary of Biological Variation for Cardiac Markers MARKERCV_(A) CV_(I) CV_(T) II RCV DURATION REFERNCE Acute disease markersCreatine kinase 14.0 22 42.2 0.52 72.2 Daily Ross et al. CK-MB, activity29.1 4.9 14.1 0.35 81.8 Daily Ross et al. CK-MB, mass 6.8 18.4 61.2 0.3054.4 Daily Ross et al. Myoglobin 13.4 17.6 46.6 0.38 61.2 Daily Ross etal. Troponin I 7.6 7.6 10.9 0.72 +35, −26 Hourly This Study Chronicdisease markers Myoglobin 6.0 11.1 13.8 0.80 35.0 Weekly Panteghini etal. C-reactive protein 5.2 42.2 92.5 0.46 118 Weekly Macy et al.C-reactive protein 1.0 36.8 62.2 0.59 102 4 days Cho et al. Serumamyloid A 4.0 25.0 61.0 0.40 70.1 Weekly Melzi d'Eril et al.Myeloperoxidase 4.0 36.0 30.0 1.20 100 Weekly Dednam et al. BNP 8.4 40.041.0 0.98 113 Weekly Wu et al. BNP +198, −66 Weekly Fokkema NT-proBNP3.0 35.0 35.0 1.00 98 Weekly Bruins et al. NT-proBNP 1.6 33.3 36.5 0.9092 Weekly Wu et al. NT-BNP +157, −61 Weekly Fokkema Troponin I 12.2 9.015.2 0.59 52, −34 Weekly This Study ¹Conducted in healthy subjects.²Conducted in stable heart failure patients. ³Lognormal transformationof Bruins et al. (22) data.

These markers include myoglobin, high sensitivity C-reactive protein(hsCRP), serum amyloid A, and myeloperoxidase for atherosclerosis, andB-type natriuretic peptide (BNP) and NT-proBNP for heart failure 23).Both the CV_(I) and CV_(T) are in general higher for long-term “disease”markers than for the short-term event markers. This is not unexpectedgiven that the interval between blood collections is weeks instead ofhours. The low index of individuality for the inflammatory markers(0.4-0.59, from Table 2) suggests that reference intervals will not beuseful, and that serial testing will be required. In contrast, for thenatriuretic peptides, the higher index of individuality (0.9-1.1, Table2) suggests that reference intervals are appropriate. For both theinflammatory markers and natriuretic peptides, the high biologicalvariation results in relatively high RCV values (70 to 198%). Thereforeminor changes in serial results will not be meaningful as recentlysuggested for hsCRP. Others have challenged the utility of biologicalvariation for determining significant serial change values for hsCRP andBNP. The results for the index of individuality for cardiac troponin arebetween hsCRP and BNP.

The results for cardiac troponin for monitoring of cardiac disease aresignificantly different than for the other chronic cardiac diseasemarkers. The biological variation, index of individuality and RCVs aremuch lower. Although hsCRP, myeloperoxidase and BNP are useful for riskstratification, the results on troponin suggest that high sensitivityassays can be applied to detection of minor myocardial damage due todrugs that may be cardiotoxic. Patients on these drugs are currently notroutinely tested for cardiac troponin. This is because the existingcommercial assays lack the sensitivity to low to detect very minor andchronic increases. With the development of high sensitive assays, theutility of cardiac troponin should be re-examined for this application.If proven to be useful, the long-term biological variation andcalculation of the RCV will be relevant. Recently, high-sensitivitytroponin T assays have been developed and similar biological variabilitystudies need to be performed to determine how serial changes can beinterpreted in the context of acute and chronic cardiovascular disease.

Example 9 Specificity of a High Sensitivity Cardiac Troponin-I AssayUtilizing Single Molecule Counting Technology

Recent guidelines for cTnI recommend a 99^(th) percentile cutoff valuewith assay imprecision ≦10%. However, most commercial assays do not meetthese criteria and cTnI values below the 99^(th)% value providediagnostic and prognostic information.

Values of cardiac troponin I are the mainstay for the diagnosis orexclusion of acute myocardial infarction (AMI) and for riskstratification in patients who present with acute coronary syndromes.The European Society of Cardiology, American Heart Association, WorldHeart Federation and American College of Cardiology have recommendedthat the 99^(th) percentile be used as the most appropriate cutoffconcentration for troponin. It is recommended that there be no more than10% assay imprecision at the 99^(th) percentile value. Given that mostcommercial assays do not have the sensitivity and precision to meetthese goals and the fact that it has become clear over time thatdetectable values below the 99^(th) percentile value carry importantprognostic information, there is interest among manufacturers toincrease the sensitivity of commercial troponin assays.

Recently, a new cardiac troponin I (cTnI) assay was described that usessingle-photon fluorescence detection and antibody coated microparticles(MPs) as a solid phase capture reagent. It appears considerably moresensitive than commercial assays. The analyzer, compositions, andmethods of the cTnI assay reports values of cardiac troponin I in theblood of healthy subjects, with a preliminary 10% CV at 1.8 ng/L and a99^(th) percentile cutoff value of 10 ng/L. These lower cutoff valueslikely will produce more and earlier positive test results in patientswho present to emergency departments with chest pain than the oldertroponin assays.

It is important to demonstrate the analytical specificity of this highlysensitive cTnI assay with regard to false-negatives and false-positives.Some degree of assay interference has already been recognized fromheparinized and chelated serum samples, as well as troponin autoantibodyinterference, which produce false-negative results at low troponinconcentrations. False-positives may occur if troponin readings fromhealthy subjects are due to non-specific binding of serum or plasmacomponents to the antibodies used in the assay, rendering results of theassay invalid. This investigation was designed to indirectly test thisissue, predicated on the assumption that if a non-specific cTnI signalwas being obtained from the samples, it would also be observed by otherantibody coated MPs.

Methods

Concentrations of cTnI were determined from 20 normal subjects acrossfour specimen types (serum, EDTA, lithium heparin and citrate plasma)and 8 additional serum samples from apparently healthy blood donors(AVG+/−SD). To test for specificity, microparticles (MPs) were coatedwith a panel of unrelated antibodies (A-beta42, MIP-1a, G-CSF and PSA)or left un-coated (blank MPs) and assayed for cTnI. Standard curves weregenerated using the NIST cTnI standard analyte compared to a skeletaltroponin analyte.

Serum, EDTA, lithium heparin and sodium citrated plasma was collectedfrom 20 subjects who were free of cardiac disease or symptoms (13female, 7 male, average 43y, range 23-64y). The protocol was reviewedand approved by the University of California Committee on Human Researchand all subjects signed a written consent. The blood was centrifugedwithin 30 minutes of collection, aliquoted and stored frozen at −70° C.until analysis. Eight additional serum samples were obtained fromhealthy blood bank donors to test for non-specific antibody interactions(4 male, 4 female, average 25.5y, range 22-30y). Frozen serum and plasmawere provided to Singulex (blinded to donor ID) and assayed with theanalyzer, compositions, and methods. Briefly, specimens were thawed atroom temperature, centrifuged for 10 minutes at 13,000×g and theclarified specimen was placed into a clean tube. Each 50 ul specimen(neat serum or plasma) was mixed with 150 ul assay buffer containingparamagnetic microparticles (Dynabeads MyOne Streptavidin C1,Invitrogen) in the well of a 96 plate. For analysis across sera types,MPs were coated with a cTnI capture monoclonal antibody (R&D Systems).For specificity studies, MPs were either left un-coated (blank MPs) orcoated with one of the following monoclonal antibodies: amyloid-beta-42(A-beta42, Covance), macrophage inflammatory protein-1 alpha (MIP-1a,R&D Systems), granulocyte-colony stimulating factor (G-CSF, R&DSystems), and prostate specific antigen (PSA, BiosPacific). Theseantibodies (both isotype matched and un-matched) were chosen torepresent a variety of antibodies raised against epitopes which areun-related to cardiac troponin I, to test for evidence of non-specificantibody interactions in the assay conditions. All antibodies werebiotinylated and coated onto streptavidin MPs at 15 ug IgG per mg of MPsaccording to manufacturer's recommendations. The assay consisted of aone hour capture step, one wash of MPs, a 30 minute detection step withpolyclonal goat anti cTnI (Biospacific) labeled with fluorescent dye anddiluted in assay buffer, 5 washes, and 30 minutes of elution ofdetection antibody bound to the MPs. All specimens, calibrators andcontrols were tested in triplicate and results are presented as anaverage of the determinations+/−standard deviation. Inter-assayprecision was determined by assay of two control sera over 14 assay runsperformed over 4 days. Analyte specificity was tested through linearregression analysis of cTnI standard (NIST) and non-specific skeletaltroponin (Hytest) analytes, assayed over a range of 0.1-100 ng/L.

Results

cTnI was quantifiable in 93% of serum and plasma specimens, and nosignificant differences in cTnI concentrations across specimen typeswere observed (95% CI). Out of all samples and non-specific antibodiestested, one sample displayed quantifiable non-specific interaction withthe A-beta-42 antibody (55% CR). No non-specific events were observedwhen skeletal troponin was used as the analyte.

From the 20 donor sera and plasma samples collected, there were noapparent trends in cTnI measurements based upon donor's age or sex (FIG.15). Figure shows concentrations of cTnI (ng/L) in matched serum andplasma specimens obtained from 20 human donors of mixed age and sexSamples were run in triplicate and results for each sample are presentedas the mean+/−standard deviation. For each donor the average cTnI valuewas assessed across the 4 specimen types (average 3.76+/−4.76 ng/L,range 0.84-22.37 ng/L). All determinations fell within two standarddeviations from the mean, indicating no significant differences in cTnIconcentration between specimen types (95% CI). One donor presented withelevated levels of cTnI across all specimen types, and this maximumvalue was identified as a statistical outlier using Grub's test (p<0.05)and removed from further analysis. Removal of this sample produced anadjusted average cTnI value of 2.78+/−1.93 ng/L similar to valuesreported previously. In two control sera, inter-assay precisions of 7%CV and 10% CV were measured with average cTnI measurements of 8.3 and2.2 ng/L, respectively.

Across all 28 donors, cTnI was detectable in all (LoD 0.2 ng/L) andquantifiable for 93% (LLoQ 1 ng/L) of matched serum and plasma specimenstested (Table 11).

TABLE 11 Cross reactivity in normal, human serum samples of the SingulexMP- based cTnI Assay using non-specific capture antibodies. DNQ ND (1.0ng/L > Quantifiable MP Total (<LoD, X ≧ (≧LLoQ, Capture Samples 0.2ng/L) 0.2 ng/L) 1.0 ng/L) Antibody (n) (n) % (n) % (n) % cTnI 28 0 0 2 726  93 Blank 28 26 93 2 7 0 0 AB-42 20 18 90 1 5  1* 5 MIP-1a 8 6 75 225 0 0 PSA 8 7 88 1 13 0 0 G-CSF 8 8 100 0 0 0 0 *One serum sampledisplayed 55% CR with AB-42 (1.26 ng/L) compared to cTnI (2.36 ng/L).Abbreviations: Cross reactivity (CR), non-detectable (ND), detectablebut non-quantifiable (DNQ)

Serum and plasma specimens from one of the twenty volunteers (5%) wasquantifiable for a non-specific antibody, which displayed 55% crossreactivity with A-beta-42 (1.26 ng/L) compared to cTnI (2.36 ng/L). Noother samples were quantifiable for cTnI when using a non-specificcapture antibody or when using blank MPs. Some low level non-specificreaction (less than the LLoQ and greater than the LoD) was observed forblank microparticles (7%) and for non-specific capture antibodiesA-beta-42 (5% donors), MIP-1a (25% donors), and PSA (13% donors). Nonon-specific reactions were observed when G-CSF was used as the captureantibody. In tests of analyte specificity, back-interpolated standardcurve analysis of the NIST standard cTnI analyte (y=1.053x−0.229,R²=0.999) showed poor correlation when compared to analysis of theorthologous skeletal troponin analyte (y=0.000x+0.156, R²=0.001) in theanalyzer, compositions, and methods.

These data support the specificity of the novel single molecule assayfor the quantification of cTnI. We have previously shown that serumspecimens provide a linear response when diluted with syntheticcalibrator diluent, however, we could not exclude the possibility thatwith such sensitive limits of detection some other material was capturedalong with cTnI and then non-specifically measured. This study providesa more robust test by substituting a series of non-specific, non-isotypematched, but high affinity antibodies in the capture position to furtherevaluate that possibility. Our data support the contention that theassay specifically measures cTnI in most apparently healthy subjects.Even in subjects that showed low-levels of non-specific binding, thisbinding was below the limit of quantification reported for the assay inall but one case (See Table 11). In the one case where non-specificbinding was quantifiable, it accounted for 55% of the cTnI signal. Themagnitude of this effect would not have moved this subject out of thenormal range projected for the assay.

Data like these are critical as the field moves forward. As moresensitive detection techniques such as the novel assay used in thisapproach are developed, confirmation of the specificity of theirdetection is essential. Thus, similar cross reactivity studies whichevaluate potential issues with troponin orthologs (i.e., skeletal muscleTnI and TnT) and other non-specific binding events are critical adjunctsto the validation of these assays. Because of the sensitivity of thisnovel assay, conventional methods may not be adequate to provide thisspecificity information, requiring an equally novel approach to thevalidation issue. These data, taken together with those reportedpreviously predicated on linear dilution and immunodepletion, providestrong evidence that this particular cTnI assay approach has maintainedthe specificity of detection.

The data presented provides further documentation that the analyzer,compositions, and methods have high specificity and reliably measurecTnI in apparently healthy human subjects. This high degree ofspecificity and high sensitivity should allow for further investigationof the clinical significance of mild increases in cTnI in patients withand without acute coronary disease.

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What is claimed is:
 1. A method for determining an absence of coronaryartery disease (CAD) in a subject, the method comprising: (a) measuringthe concentration of at least one of Troponin I (cTnI) and cardiactroponin T (cTnT) in a blood, serum or plasma sample from the subject inan assay comprising counting individual digital events representingbinding of a binding partner the at least one of cTnI and cTnT, whereineach individual digital event comprises a signal representing a singlemolecule of the cTnI and cTnT in the sample, determining a total signalas a sum of the individual digital events, and relating the sum to theconcentration of the at least one of cTnI and cTnT in the sample, (b)determining an absence of CAD in the subject when the concentration ofthe at least one of cTnI and cTnT in the sample is less than about 10pg/mL.
 2. The method of claim 1, wherein the subject has a symptom ofcoronary artery disease.
 3. The method of claim 2, wherein the symptomis at least one of chest pain, shortness of breath, arm pain, abnormalEKG, and abnormal enzyme levels.
 4. The method of claim 1, wherein theCAD is selected from at least one of acute myocardial infarction,cardiac damage, cardiac toxicity, acute coronary syndrome, myocarditis,and heart failure.
 5. The method of claim 1, wherein the absence of CADis determined when the at least one of cTnI and cTnT in the sample isless than about 9 pg/mL.
 6. The method of claim 1, wherein the absenceof CAD is determined when the at least one of cTnI and cTnT in thesample is less than about 8 pg/mL.
 7. The method of claim 1, wherein theabsence of CAD is determined when the at least one of cTnI and cTnT inthe sample is less than about 7 pg/mL.
 8. The method of claim 1, whereinthe sample is taken during a stress test.
 9. The method of claim 1,wherein the sample is taken after a stress test.
 10. The method of claim1, wherein the assay has a limit of detection for the at least one ofcTnI and cTnT of less than 3 pg/mL and a coefficient of variation ofless than 10%.
 11. The method of claim 1, wherein the binding partnercomprises an antibody specific for the at least one of cTnI and cTnT.12. The method of claim 11, wherein the assay detects a photon emissionfrom a label corresponding to a single molecule of the at least one ofcTnI and cTnT in the sample, wherein the antibody is bound to or wasbound to a molecule of the at least one of cTnI and cTnT in the sample.13. The method of claim 12, wherein the antibody is eluted from the atleast one of cTnI or cTnT before the photon emission is detected. 14.The method of claim 13, wherein the photon emission is detected in asingle molecule analyzer.
 15. The method of claim 1, wherein the assayis conducted using an analyzer comprising: (a) an electromagneticradiation source for stimulating a fluorescent moiety; and (b) anelectromagnetic radiation detector that measures an electromagneticcharacteristic of the fluorescent moiety when stimulated by theelectromagnetic radiation.
 16. The method of claim 1, wherein the atleast one of Cardiac Troponin I (cTnI) and cardiac troponin T (cTnT) iscTnI.
 17. The method of claim 1, wherein the at least one of CardiacTroponin I (cTnI) and cardiac troponin T (cTnT) is cTnT.
 18. A methodfor determining an absence of coronary artery disease (CAD) in asubject, comprising: (a) measuring the concentration of at least one ofTroponin I (cTnI) and cardiac troponin T (cTnT) in a series of blood,serum or plasma samples from the subject in an assay comprising countingindividual digital events representing binding of a binding partner tothe at least one of cTnI and cTnT, wherein each individual digital eventcomprises a signal representing a single molecule of the cTnI and cTnTin the sample, determining a total signal as a sum of the individualdigital events, and relating the sum to the concentration of the atleast one of cTnI and cTnT in the sample, (b) determining at least oneof a change and a rate of change in the concentration of the at leastone of cTnI and cTnT in the series of samples, (c) determining theabsence of coronary artery disease when the at least one of the changeand the rate of change of the at least one of cTnI and cTnT in theseries of samples is less than a predetermined reference change inconcentration of less than 1-10 pg/mL or a predetermined reference rateof change in concentration of 2-10 pg/mL.
 19. The method of claim 18,wherein the assay has a limit of detection of less than 3 pg/mL for theat least one of cTnI and cTnT.
 20. The method of claim 18, wherein thecoronary artery disease is at least one of acute myocardial infarction,cardiac damage, cardiac toxicity, acute coronary syndrome, myocarditis,and heart failure.
 21. The method of claim 18, wherein the subject has asymptom of coronary artery disease.
 22. The method of claim 21, whereinthe symptom is at least one of abnormal EKG, abnormal enzyme levels,chest pain, arm pain, and shortness of breath.
 23. The method of claim18, further comprising comparing the concentration of the at least oneof cTnI and cTnT to a threshold concentration representing the 99thpercentile concentration of the at least of one cTnI and cTnT in a groupof normal subjects.
 24. The method of claim 23, wherein the thresholdconcentration is less than 10 pg/mL.
 25. The method of claim 23, whereinthe threshold concentration is less than about 7 pg/ml.
 26. The methodof claim 23, wherein the threshold concentration is less than about 8pg/ml.
 27. The method of claim 23, wherein the threshold concentrationis less than about 9 pg/ml.
 28. The method of claim 23, wherein thethreshold concentration is within a range of 1 pg/ml to 10 pg/ml. 29.The method of claim 18, wherein at least one sample of the series ofsamples is taken during or after a cardiac stress test.
 30. The methodof claim 18, wherein the series of samples is taken over a period of upto about 30 hours.
 31. The method of claim 30, wherein the series ofsamples is taken at intervals of less than about 4 hours.
 32. The methodof claim 18, wherein the binding partner comprises an antibody specificfor the at least one of cTnI and cTnT.
 33. The method of claim 32,wherein the assay detects a photon emission from a label correspondingto a single molecule of the at least one of cTnI and cTnT in the sample,wherein the antibody is bound to or was bound to a molecule of the atleast one of cTnI and cTnT in the sample.
 34. The method of claim 33,wherein the antibody is eluted from the at least one of cTnI or cTnTbefore the photon event.
 35. The method of claim 33, wherein the photonemission is detected in a single molecule analyzer.
 36. The method ofclaim 18, wherein the assay is conducted using an analyzer comprising:(a) an electromagnetic radiation source for stimulating a fluorescentmoiety; and (b) an electromagnetic radiation detector that measures anelectromagnetic characteristic of the fluorescent moiety when stimulatedby the electromagnetic radiation.
 37. The method of claim 18, whereinthe at least one of Cardiac Troponin I (cTnI) and cardiac troponin T(cTnT) is cTnI.
 38. The method of claim 18, wherein the at least one ofCardiac Troponin I (cTnI) and cardiac troponin T (cTnT) is cTnT.
 39. Themethod of claim 1, wherein the assay has a sensitivity for the at leastone of cTnI and cTnT of less than 3 pg/mL.
 40. The method of claim 1,wherein the assay has a sensitivity for the at least one of cTnI andcTnT of less than 1 pg/mL.
 41. The method of claim 18, wherein the assayhas a sensitivity for the at least one of cTnI and cTnT of less than 3pg/mL.
 42. The method of claim 18, wherein the assay has a sensitivityfor the at least one of cTnI and cTnT of less than 1 pg/mL.
 43. Themethod of claim 39, wherein the at least one of Cardiac Troponin I(cTnI) and cardiac troponin T (cTnT) is cTnI.
 44. The method of claim40, wherein the at least one of Cardiac Troponin I (cTnI) and cardiactroponin T (cTnT) is cTnI.
 45. The method of claim 41, wherein the atleast one of Cardiac Troponin I (cTnI) and cardiac troponin T (cTnT) iscTnI.
 46. The method of claim 42, wherein the at least one of CardiacTroponin I (cTnI) and cardiac troponin T (cTnT) is cTnI.
 47. The methodof claim 2, wherein the at least one of Cardiac Troponin I (cTnI) andcardiac troponin T (cTnT) is cTnI.
 48. The method of claim 5, whereinthe at least one of Cardiac Troponin I (cTnI) and cardiac troponin T(cTnT) is cTnI.
 49. The method of claim 6, wherein the at least one ofCardiac Troponin I (cTnI) and cardiac troponin T (cTnT) is cTnI.
 50. Themethod of claim 7, wherein the at least one of Cardiac Troponin I (cTnI)and cardiac troponin T (cTnT) is cTnI.
 51. The method of claim 10,wherein the at least one of Cardiac Troponin I (cTnI) and cardiactroponin T (cTnT) is cTnI.
 52. The method of claim 12, wherein the atleast one of Cardiac Troponin I (cTnI) and cardiac troponin T (cTnT) iscTnI.
 53. The method of claim 19, wherein the at least one of CardiacTroponin I (cTnI) and cardiac troponin T (cTnT) is cTnI.
 54. The methodof claim 23, wherein the at least one of Cardiac Troponin I (cTnI) andcardiac troponin T (cTnT) is cTnI.
 55. The method of claim 24, whereinthe at least one of Cardiac Troponin I (cTnI) and cardiac troponin T(cTnT) is cTnI.
 56. The method of claim 25, wherein the at least one ofCardiac Troponin I (cTnI) and cardiac troponin T (cTnT) is cTnI.
 57. Themethod of claim 26, wherein the at least one of Cardiac Troponin I(cTnI) and cardiac troponin T (cTnT) is cTnI.
 58. The method of claim27, wherein the at least one of Cardiac Troponin I (cTnI) and cardiactroponin T (cTnT) is cTnI.
 59. The method of claim 28, wherein the atleast one of Cardiac Troponin I (cTnI) and cardiac troponin T (cTnT) iscTnI.